CN112870336B - 一种白细胞介素29突变体蛋白制剂 - Google Patents
一种白细胞介素29突变体蛋白制剂 Download PDFInfo
- Publication number
- CN112870336B CN112870336B CN202110206470.3A CN202110206470A CN112870336B CN 112870336 B CN112870336 B CN 112870336B CN 202110206470 A CN202110206470 A CN 202110206470A CN 112870336 B CN112870336 B CN 112870336B
- Authority
- CN
- China
- Prior art keywords
- leu
- ala
- seq
- mutant protein
- pro
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 101001002470 Homo sapiens Interferon lambda-1 Proteins 0.000 title claims abstract description 171
- 102100020990 Interferon lambda-1 Human genes 0.000 title claims abstract description 171
- 108010021466 Mutant Proteins Proteins 0.000 title claims abstract description 86
- 102000008300 Mutant Proteins Human genes 0.000 title claims abstract description 86
- 238000002360 preparation method Methods 0.000 title claims abstract description 53
- 239000007853 buffer solution Substances 0.000 claims abstract description 28
- 229910021645 metal ion Inorganic materials 0.000 claims abstract description 11
- 239000002738 chelating agent Substances 0.000 claims abstract description 10
- 239000000203 mixture Substances 0.000 claims description 57
- 238000009472 formulation Methods 0.000 claims description 54
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 36
- 229940124274 edetate disodium Drugs 0.000 claims description 24
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 21
- 239000011780 sodium chloride Substances 0.000 claims description 18
- 235000001014 amino acid Nutrition 0.000 claims description 16
- 150000001413 amino acids Chemical group 0.000 claims description 15
- 239000008363 phosphate buffer Substances 0.000 claims description 15
- 239000008055 phosphate buffer solution Substances 0.000 claims description 14
- 239000004471 Glycine Substances 0.000 claims description 11
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 11
- 235000003704 aspartic acid Nutrition 0.000 claims description 11
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 11
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Chemical group OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims description 9
- 235000004400 serine Nutrition 0.000 claims description 9
- 239000000126 substance Substances 0.000 claims description 7
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 6
- 230000003204 osmotic effect Effects 0.000 claims description 6
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical group OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 claims description 5
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical group C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims description 5
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Chemical group CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 5
- 239000004473 Threonine Chemical group 0.000 claims description 5
- -1 aliphatic amino acid Chemical class 0.000 claims description 5
- 235000018417 cysteine Nutrition 0.000 claims description 5
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 5
- 235000013922 glutamic acid Nutrition 0.000 claims description 5
- 239000004220 glutamic acid Substances 0.000 claims description 5
- 235000008521 threonine Nutrition 0.000 claims description 5
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 claims description 4
- 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 4
- 239000012535 impurity Substances 0.000 abstract description 15
- 238000000034 method Methods 0.000 abstract description 12
- 238000006731 degradation reaction Methods 0.000 abstract description 4
- 238000003860 storage Methods 0.000 abstract description 4
- 230000015556 catabolic process Effects 0.000 abstract description 3
- 230000002776 aggregation Effects 0.000 abstract description 2
- 238000004220 aggregation Methods 0.000 abstract description 2
- 230000008569 process Effects 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 description 58
- 102000004169 proteins and genes Human genes 0.000 description 49
- 235000018102 proteins Nutrition 0.000 description 48
- 239000000523 sample Substances 0.000 description 36
- OVBJJZOQPCKUOR-UHFFFAOYSA-L EDTA disodium salt dihydrate Chemical group O.O.[Na+].[Na+].[O-]C(=O)C[NH+](CC([O-])=O)CC[NH+](CC([O-])=O)CC([O-])=O OVBJJZOQPCKUOR-UHFFFAOYSA-L 0.000 description 33
- 238000002474 experimental method Methods 0.000 description 26
- 239000000243 solution Substances 0.000 description 22
- 238000001514 detection method Methods 0.000 description 21
- 239000003814 drug Substances 0.000 description 20
- 125000003275 alpha amino acid group Chemical group 0.000 description 17
- 210000004027 cell Anatomy 0.000 description 17
- 238000005342 ion exchange Methods 0.000 description 17
- 238000005259 measurement Methods 0.000 description 17
- 229940079593 drug Drugs 0.000 description 16
- 239000007788 liquid Substances 0.000 description 16
- 239000012071 phase Substances 0.000 description 16
- 238000012360 testing method Methods 0.000 description 16
- 239000000872 buffer Substances 0.000 description 15
- 238000004007 reversed phase HPLC Methods 0.000 description 15
- 229940024606 amino acid Drugs 0.000 description 14
- 238000001727 in vivo Methods 0.000 description 13
- 230000004071 biological effect Effects 0.000 description 12
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 12
- 230000000694 effects Effects 0.000 description 12
- 230000035772 mutation Effects 0.000 description 11
- WKPXXXUSUHAXDE-SRVKXCTJSA-N Arg-Pro-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O WKPXXXUSUHAXDE-SRVKXCTJSA-N 0.000 description 10
- OVPYIUNCVSOVNF-ZPFDUUQYSA-N Ile-Gln-Pro Natural products CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(O)=O OVPYIUNCVSOVNF-ZPFDUUQYSA-N 0.000 description 10
- SBANPBVRHYIMRR-UHFFFAOYSA-N Leu-Ser-Pro Natural products CC(C)CC(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O SBANPBVRHYIMRR-UHFFFAOYSA-N 0.000 description 10
- 241000699670 Mus sp. Species 0.000 description 10
- 108010049041 glutamylalanine Proteins 0.000 description 10
- JYPCXBJRLBHWME-UHFFFAOYSA-N glycyl-L-prolyl-L-arginine Natural products NCC(=O)N1CCCC1C(=O)NC(CCCN=C(N)N)C(O)=O JYPCXBJRLBHWME-UHFFFAOYSA-N 0.000 description 10
- OVSKVOOUFAKODB-UWVGGRQHSA-N Gly-Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N OVSKVOOUFAKODB-UWVGGRQHSA-N 0.000 description 9
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 9
- JNDYEOUZBLOVOF-AVGNSLFASA-N Leu-Leu-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O JNDYEOUZBLOVOF-AVGNSLFASA-N 0.000 description 9
- 238000003556 assay Methods 0.000 description 9
- 238000000889 atomisation Methods 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 8
- XWUIHCZETFNRPA-IHPCNDPISA-N His-His-Trp Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(O)=O)C1=CN=CN1 XWUIHCZETFNRPA-IHPCNDPISA-N 0.000 description 8
- PJYSOYLLTJKZHC-GUBZILKMSA-N Leu-Asp-Gln Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCC(N)=O PJYSOYLLTJKZHC-GUBZILKMSA-N 0.000 description 8
- DZQMXBALGUHGJT-GUBZILKMSA-N Leu-Glu-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O DZQMXBALGUHGJT-GUBZILKMSA-N 0.000 description 8
- VZBIUJURDLFFOE-IHRRRGAJSA-N Leu-His-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O VZBIUJURDLFFOE-IHRRRGAJSA-N 0.000 description 8
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 8
- FYPGHGXAOZTOBO-IHRRRGAJSA-N Pro-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@@H]2CCCN2 FYPGHGXAOZTOBO-IHRRRGAJSA-N 0.000 description 8
- KKHRWGYHBZORMQ-NHCYSSNCSA-N Val-Arg-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N KKHRWGYHBZORMQ-NHCYSSNCSA-N 0.000 description 8
- 108010001271 arginyl-glutamyl-arginine Proteins 0.000 description 8
- 210000004072 lung Anatomy 0.000 description 8
- 108010054155 lysyllysine Proteins 0.000 description 8
- 230000009467 reduction Effects 0.000 description 8
- 108010080629 tryptophan-leucine Proteins 0.000 description 8
- RLMISHABBKUNFO-WHFBIAKZSA-N Ala-Ala-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O RLMISHABBKUNFO-WHFBIAKZSA-N 0.000 description 7
- SSSROGPPPVTHLX-FXQIFTODSA-N Ala-Arg-Asp Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O SSSROGPPPVTHLX-FXQIFTODSA-N 0.000 description 7
- NJIFPLAJSVUQOZ-JBDRJPRFSA-N Ala-Cys-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](C)N NJIFPLAJSVUQOZ-JBDRJPRFSA-N 0.000 description 7
- LJFNNUBZSZCZFN-WHFBIAKZSA-N Ala-Gly-Cys Chemical compound N[C@@H](C)C(=O)NCC(=O)N[C@@H](CS)C(=O)O LJFNNUBZSZCZFN-WHFBIAKZSA-N 0.000 description 7
- BTRULDJUUVGRNE-DCAQKATOSA-N Ala-Pro-Lys Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(O)=O BTRULDJUUVGRNE-DCAQKATOSA-N 0.000 description 7
- BIGRHVNFFJTHEB-UBHSHLNASA-N Asn-Trp-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(O)=O)C(O)=O BIGRHVNFFJTHEB-UBHSHLNASA-N 0.000 description 7
- WZZGXXNRSZIQFC-VGDYDELISA-N Cys-His-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CS)N WZZGXXNRSZIQFC-VGDYDELISA-N 0.000 description 7
- LGIKBBLQVSWUGK-DCAQKATOSA-N Gln-Leu-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O LGIKBBLQVSWUGK-DCAQKATOSA-N 0.000 description 7
- PBYFVIQRFLNQCO-GUBZILKMSA-N Gln-Pro-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O PBYFVIQRFLNQCO-GUBZILKMSA-N 0.000 description 7
- WATXSTJXNBOHKD-LAEOZQHASA-N Glu-Asp-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O WATXSTJXNBOHKD-LAEOZQHASA-N 0.000 description 7
- KRRMJKMGWWXWDW-STQMWFEESA-N Gly-Arg-Phe Chemical compound NC(=N)NCCC[C@H](NC(=O)CN)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 KRRMJKMGWWXWDW-STQMWFEESA-N 0.000 description 7
- PDUHNKAFQXQNLH-ZETCQYMHSA-N Gly-Lys-Gly Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)NCC(O)=O PDUHNKAFQXQNLH-ZETCQYMHSA-N 0.000 description 7
- GVKKVHNRTUFCCE-BJDJZHNGSA-N Ile-Leu-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)O)N GVKKVHNRTUFCCE-BJDJZHNGSA-N 0.000 description 7
- BQSLGJHIAGOZCD-CIUDSAMLSA-N Leu-Ala-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O BQSLGJHIAGOZCD-CIUDSAMLSA-N 0.000 description 7
- STAVRDQLZOTNKJ-RHYQMDGZSA-N Leu-Arg-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O STAVRDQLZOTNKJ-RHYQMDGZSA-N 0.000 description 7
- ZTLGVASZOIKNIX-DCAQKATOSA-N Leu-Gln-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ZTLGVASZOIKNIX-DCAQKATOSA-N 0.000 description 7
- WIDZHJTYKYBLSR-DCAQKATOSA-N Leu-Glu-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O WIDZHJTYKYBLSR-DCAQKATOSA-N 0.000 description 7
- AOFYPTOHESIBFZ-KKUMJFAQSA-N Leu-His-His Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O AOFYPTOHESIBFZ-KKUMJFAQSA-N 0.000 description 7
- WXUOJXIGOPMDJM-SRVKXCTJSA-N Leu-Lys-Asn Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O WXUOJXIGOPMDJM-SRVKXCTJSA-N 0.000 description 7
- VEGLGAOVLFODGC-GUBZILKMSA-N Lys-Glu-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O VEGLGAOVLFODGC-GUBZILKMSA-N 0.000 description 7
- IRVONVRHHJXWTK-RWMBFGLXSA-N Met-Lys-Pro Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N1CCC[C@@H]1C(=O)O)N IRVONVRHHJXWTK-RWMBFGLXSA-N 0.000 description 7
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 7
- PEFJUUYFEGBXFA-BZSNNMDCSA-N Phe-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC1=CC=CC=C1 PEFJUUYFEGBXFA-BZSNNMDCSA-N 0.000 description 7
- MMJJFXWMCMJMQA-STQMWFEESA-N Phe-Pro-Gly Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)NCC(O)=O)C1=CC=CC=C1 MMJJFXWMCMJMQA-STQMWFEESA-N 0.000 description 7
- IFMDQWDAJUMMJC-DCAQKATOSA-N Pro-Ala-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O IFMDQWDAJUMMJC-DCAQKATOSA-N 0.000 description 7
- UPJGUQPLYWTISV-GUBZILKMSA-N Pro-Gln-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O UPJGUQPLYWTISV-GUBZILKMSA-N 0.000 description 7
- LXVLKXPFIDDHJG-CIUDSAMLSA-N Pro-Glu-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O LXVLKXPFIDDHJG-CIUDSAMLSA-N 0.000 description 7
- UBRMZSHOOIVJPW-SRVKXCTJSA-N Ser-Leu-Lys Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O UBRMZSHOOIVJPW-SRVKXCTJSA-N 0.000 description 7
- YUJLIIRMIAGMCQ-CIUDSAMLSA-N Ser-Leu-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YUJLIIRMIAGMCQ-CIUDSAMLSA-N 0.000 description 7
- CUXJENOFJXOSOZ-BIIVOSGPSA-N Ser-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CO)N)C(=O)O CUXJENOFJXOSOZ-BIIVOSGPSA-N 0.000 description 7
- QNBVFKZSSRYNFX-CUJWVEQBSA-N Ser-Thr-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CO)N)O QNBVFKZSSRYNFX-CUJWVEQBSA-N 0.000 description 7
- TYVAWPFQYFPSBR-BFHQHQDPSA-N Thr-Ala-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)NCC(O)=O TYVAWPFQYFPSBR-BFHQHQDPSA-N 0.000 description 7
- COYHRQWNJDJCNA-NUJDXYNKSA-N Thr-Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O COYHRQWNJDJCNA-NUJDXYNKSA-N 0.000 description 7
- IQIRAJGHFRVFEL-UBHSHLNASA-N Trp-Ser-Cys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N IQIRAJGHFRVFEL-UBHSHLNASA-N 0.000 description 7
- 239000008351 acetate buffer Substances 0.000 description 7
- 239000000443 aerosol Substances 0.000 description 7
- 235000014304 histidine Nutrition 0.000 description 7
- 108010085325 histidylproline Proteins 0.000 description 7
- 208000015181 infectious disease Diseases 0.000 description 7
- 108010022588 methionyl-lysyl-proline Proteins 0.000 description 7
- 238000006467 substitution reaction Methods 0.000 description 7
- 108010061238 threonyl-glycine Proteins 0.000 description 7
- 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 description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- 102000014150 Interferons Human genes 0.000 description 6
- 108010050904 Interferons Proteins 0.000 description 6
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 6
- 238000011081 inoculation Methods 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 238000013094 purity test Methods 0.000 description 6
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 5
- MQIGTEQXYCRLGK-BQBZGAKWSA-N Ala-Gly-Pro Chemical compound C[C@H](N)C(=O)NCC(=O)N1CCC[C@H]1C(O)=O MQIGTEQXYCRLGK-BQBZGAKWSA-N 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 description 5
- 230000001133 acceleration Effects 0.000 description 5
- 238000000502 dialysis Methods 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 238000011068 loading method Methods 0.000 description 5
- 230000003647 oxidation Effects 0.000 description 5
- 238000007254 oxidation reaction Methods 0.000 description 5
- 229960000329 ribavirin Drugs 0.000 description 5
- HZCAHMRRMINHDJ-DBRKOABJSA-N ribavirin Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1N=CN=C1 HZCAHMRRMINHDJ-DBRKOABJSA-N 0.000 description 5
- 238000005070 sampling Methods 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- DJIMLSXHXKWADV-CIUDSAMLSA-N Asn-Leu-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(N)=O DJIMLSXHXKWADV-CIUDSAMLSA-N 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 108010014726 Interferon Type I Proteins 0.000 description 4
- 102000002227 Interferon Type I Human genes 0.000 description 4
- 241000725643 Respiratory syncytial virus Species 0.000 description 4
- 241000144282 Sigmodon Species 0.000 description 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 239000008186 active pharmaceutical agent Substances 0.000 description 4
- 230000000840 anti-viral effect Effects 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000005286 illumination Methods 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 210000003000 inclusion body Anatomy 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 229930027917 kanamycin Natural products 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 108010034529 leucyl-lysine Proteins 0.000 description 4
- 229930182817 methionine Natural products 0.000 description 4
- 238000010172 mouse model Methods 0.000 description 4
- 210000001236 prokaryotic cell Anatomy 0.000 description 4
- 230000009385 viral infection Effects 0.000 description 4
- CCDFBRZVTDDJNM-GUBZILKMSA-N Ala-Leu-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O CCDFBRZVTDDJNM-GUBZILKMSA-N 0.000 description 3
- UHGUKCOQUNPSKK-CIUDSAMLSA-N Asn-Leu-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)N)N UHGUKCOQUNPSKK-CIUDSAMLSA-N 0.000 description 3
- JUWZKMBALYLZCK-WHFBIAKZSA-N Asp-Gly-Asn Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O JUWZKMBALYLZCK-WHFBIAKZSA-N 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- MCAVASRGVBVPMX-FXQIFTODSA-N Gln-Glu-Ala Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O MCAVASRGVBVPMX-FXQIFTODSA-N 0.000 description 3
- PNENQZWRFMUZOM-DCAQKATOSA-N Gln-Glu-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O PNENQZWRFMUZOM-DCAQKATOSA-N 0.000 description 3
- IQACOVZVOMVILH-FXQIFTODSA-N Glu-Glu-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O IQACOVZVOMVILH-FXQIFTODSA-N 0.000 description 3
- 108010074328 Interferon-gamma Proteins 0.000 description 3
- 102000008070 Interferon-gamma Human genes 0.000 description 3
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 3
- PPBKJAQJAUHZKX-SRVKXCTJSA-N Leu-Cys-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@H](C(O)=O)CC(C)C PPBKJAQJAUHZKX-SRVKXCTJSA-N 0.000 description 3
- BGZCJDGBBUUBHA-KKUMJFAQSA-N Leu-Lys-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O BGZCJDGBBUUBHA-KKUMJFAQSA-N 0.000 description 3
- SBANPBVRHYIMRR-GARJFASQSA-N Leu-Ser-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N SBANPBVRHYIMRR-GARJFASQSA-N 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- UUHXBJHVTVGSKM-BQBZGAKWSA-N Pro-Gly-Asn Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O UUHXBJHVTVGSKM-BQBZGAKWSA-N 0.000 description 3
- DWGFLKQSGRUQTI-IHRRRGAJSA-N Pro-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H]1CCCN1 DWGFLKQSGRUQTI-IHRRRGAJSA-N 0.000 description 3
- 206010061603 Respiratory syncytial virus infection Diseases 0.000 description 3
- 241000144290 Sigmodon hispidus Species 0.000 description 3
- GTNCSPKYWCJZAC-XIRDDKMYSA-N Trp-Asp-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N GTNCSPKYWCJZAC-XIRDDKMYSA-N 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 210000000424 bronchial epithelial cell Anatomy 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 229940079322 interferon Drugs 0.000 description 3
- 229940047124 interferons Drugs 0.000 description 3
- 238000004255 ion exchange chromatography 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
- 239000013641 positive control Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 230000002685 pulmonary effect Effects 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 230000010076 replication Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- KSHJXDWYTZJUEI-UHFFFAOYSA-N 1-cyclopropyl-3-[[1-(4-hydroxybutyl)benzimidazol-2-yl]methyl]imidazo[4,5-c]pyridin-2-one Chemical compound N=1C2=CC=CC=C2N(CCCCO)C=1CN(C1=O)C2=CN=CC=C2N1C1CC1 KSHJXDWYTZJUEI-UHFFFAOYSA-N 0.000 description 2
- NHCPCLJZRSIDHS-ZLUOBGJFSA-N Ala-Asp-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O NHCPCLJZRSIDHS-ZLUOBGJFSA-N 0.000 description 2
- PAIHPOGPJVUFJY-WDSKDSINSA-N Ala-Glu-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O PAIHPOGPJVUFJY-WDSKDSINSA-N 0.000 description 2
- RTZCUEHYUQZIDE-WHFBIAKZSA-N Ala-Ser-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O RTZCUEHYUQZIDE-WHFBIAKZSA-N 0.000 description 2
- PEEYDECOOVQKRZ-DLOVCJGASA-N Ala-Ser-Phe Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O PEEYDECOOVQKRZ-DLOVCJGASA-N 0.000 description 2
- ARHJJAAWNWOACN-FXQIFTODSA-N Ala-Ser-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O ARHJJAAWNWOACN-FXQIFTODSA-N 0.000 description 2
- INOIAEUXVVNJKA-XGEHTFHBSA-N Arg-Thr-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O INOIAEUXVVNJKA-XGEHTFHBSA-N 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- PBVLJOIPOGUQQP-CIUDSAMLSA-N Asp-Ala-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O PBVLJOIPOGUQQP-CIUDSAMLSA-N 0.000 description 2
- CJUKAWUWBZCTDQ-SRVKXCTJSA-N Asp-Leu-Lys Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O CJUKAWUWBZCTDQ-SRVKXCTJSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- XLLSMEFANRROJE-GUBZILKMSA-N Cys-Leu-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CS)N XLLSMEFANRROJE-GUBZILKMSA-N 0.000 description 2
- YNJBLTDKTMKEET-ZLUOBGJFSA-N Cys-Ser-Ser Chemical compound SC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O YNJBLTDKTMKEET-ZLUOBGJFSA-N 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- UWMDGPFFTKDUIY-HJGDQZAQSA-N Gln-Pro-Thr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O UWMDGPFFTKDUIY-HJGDQZAQSA-N 0.000 description 2
- BPLNJYHNAJVLRT-ACZMJKKPSA-N Glu-Ser-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O BPLNJYHNAJVLRT-ACZMJKKPSA-N 0.000 description 2
- SYMSVYVUSPSAAO-IHRRRGAJSA-N His-Arg-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O SYMSVYVUSPSAAO-IHRRRGAJSA-N 0.000 description 2
- SYIPVNMWBZXKMU-HJPIBITLSA-N His-His-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CC2=CN=CN2)N SYIPVNMWBZXKMU-HJPIBITLSA-N 0.000 description 2
- PYNPBMCLAKTHJL-SRVKXCTJSA-N His-Pro-Glu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O PYNPBMCLAKTHJL-SRVKXCTJSA-N 0.000 description 2
- LNVILFYCPVOHPV-IHPCNDPISA-N His-Trp-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(C)C)C(O)=O LNVILFYCPVOHPV-IHPCNDPISA-N 0.000 description 2
- 241000711911 Human respiratory syncytial virus A2 Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- OVPYIUNCVSOVNF-KQXIARHKSA-N Ile-Gln-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N OVPYIUNCVSOVNF-KQXIARHKSA-N 0.000 description 2
- NHJKZMDIMMTVCK-QXEWZRGKSA-N Ile-Gly-Arg Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N NHJKZMDIMMTVCK-QXEWZRGKSA-N 0.000 description 2
- 102100020989 Interferon lambda-2 Human genes 0.000 description 2
- 101710099622 Interferon lambda-2 Proteins 0.000 description 2
- 102100020992 Interferon lambda-3 Human genes 0.000 description 2
- 101710099621 Interferon lambda-3 Proteins 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- VQPPIMUZCZCOIL-GUBZILKMSA-N Leu-Gln-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O VQPPIMUZCZCOIL-GUBZILKMSA-N 0.000 description 2
- GCXGCIYIHXSKAY-ULQDDVLXSA-N Leu-Phe-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O GCXGCIYIHXSKAY-ULQDDVLXSA-N 0.000 description 2
- AKVBOOKXVAMKSS-GUBZILKMSA-N Leu-Ser-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(O)=O AKVBOOKXVAMKSS-GUBZILKMSA-N 0.000 description 2
- RVOMPSJXSRPFJT-DCAQKATOSA-N Lys-Ala-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O RVOMPSJXSRPFJT-DCAQKATOSA-N 0.000 description 2
- JBRWKVANRYPCAF-XIRDDKMYSA-N Lys-Asn-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCCN)N JBRWKVANRYPCAF-XIRDDKMYSA-N 0.000 description 2
- DKTNGXVSCZULPO-YUMQZZPRSA-N Lys-Gly-Cys Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CS)C(O)=O DKTNGXVSCZULPO-YUMQZZPRSA-N 0.000 description 2
- LOGFVTREOLYCPF-RHYQMDGZSA-N Lys-Pro-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCCN LOGFVTREOLYCPF-RHYQMDGZSA-N 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- SCKXGHWQPPURGT-KKUMJFAQSA-N Phe-Lys-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O SCKXGHWQPPURGT-KKUMJFAQSA-N 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- VDHGTOHMHHQSKG-JYJNAYRXSA-N Pro-Val-Phe Chemical compound CC(C)[C@H](NC(=O)[C@@H]1CCCN1)C(=O)N[C@@H](Cc1ccccc1)C(O)=O VDHGTOHMHHQSKG-JYJNAYRXSA-N 0.000 description 2
- 108010076504 Protein Sorting Signals Proteins 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- KZURUCDWKDEAFZ-XVSYOHENSA-N Thr-Phe-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)O KZURUCDWKDEAFZ-XVSYOHENSA-N 0.000 description 2
- UQCNIMDPYICBTR-KYNKHSRBSA-N Thr-Thr-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O UQCNIMDPYICBTR-KYNKHSRBSA-N 0.000 description 2
- RGJZPXFZIUUQDN-BPNCWPANSA-N Tyr-Val-Ala Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O RGJZPXFZIUUQDN-BPNCWPANSA-N 0.000 description 2
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 2
- 108010047495 alanylglycine Proteins 0.000 description 2
- 235000009697 arginine Nutrition 0.000 description 2
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 230000024245 cell differentiation Effects 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 239000002552 dosage form Substances 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 239000010419 fine particle Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- 150000002411 histidines Chemical class 0.000 description 2
- 108010028295 histidylhistidine Proteins 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 108010018844 interferon type III Proteins 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 108010047926 leucyl-lysyl-tyrosine Proteins 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000002663 nebulization Methods 0.000 description 2
- 231100000344 non-irritating Toxicity 0.000 description 2
- 238000010606 normalization Methods 0.000 description 2
- 230000009965 odorless effect Effects 0.000 description 2
- 229960001412 pentobarbital Drugs 0.000 description 2
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000013074 reference sample Substances 0.000 description 2
- 239000013558 reference substance Substances 0.000 description 2
- 238000004153 renaturation Methods 0.000 description 2
- 210000002345 respiratory system Anatomy 0.000 description 2
- 108010026333 seryl-proline Proteins 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- BBMHARZCALWXSL-UHFFFAOYSA-M sodium dihydrogenphosphate monohydrate Chemical compound O.[Na+].OP(O)([O-])=O BBMHARZCALWXSL-UHFFFAOYSA-M 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 230000009967 tasteless effect Effects 0.000 description 2
- 108010072986 threonyl-seryl-lysine Proteins 0.000 description 2
- 210000003437 trachea Anatomy 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- HBZBAMXERPYTFS-SECBINFHSA-N (4S)-2-(6,7-dihydro-5H-pyrrolo[3,2-f][1,3]benzothiazol-2-yl)-4,5-dihydro-1,3-thiazole-4-carboxylic acid Chemical compound OC(=O)[C@H]1CSC(=N1)c1nc2cc3CCNc3cc2s1 HBZBAMXERPYTFS-SECBINFHSA-N 0.000 description 1
- KEQXNNJHMWSZHK-UHFFFAOYSA-L 1,3,2,4$l^{2}-dioxathiaplumbetane 2,2-dioxide Chemical compound [Pb+2].[O-]S([O-])(=O)=O KEQXNNJHMWSZHK-UHFFFAOYSA-L 0.000 description 1
- KIUYPHAMDKDICO-WHFBIAKZSA-N Ala-Asp-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O KIUYPHAMDKDICO-WHFBIAKZSA-N 0.000 description 1
- HXNNRBHASOSVPG-GUBZILKMSA-N Ala-Glu-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O HXNNRBHASOSVPG-GUBZILKMSA-N 0.000 description 1
- MEFILNJXAVSUTO-JXUBOQSCSA-N Ala-Leu-Thr Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MEFILNJXAVSUTO-JXUBOQSCSA-N 0.000 description 1
- RWCLSUOSKWTXLA-FXQIFTODSA-N Arg-Asp-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O RWCLSUOSKWTXLA-FXQIFTODSA-N 0.000 description 1
- WMEVEPXNCMKNGH-IHRRRGAJSA-N Arg-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N WMEVEPXNCMKNGH-IHRRRGAJSA-N 0.000 description 1
- KZXPVYVSHUJCEO-ULQDDVLXSA-N Arg-Phe-Lys Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(O)=O)CC1=CC=CC=C1 KZXPVYVSHUJCEO-ULQDDVLXSA-N 0.000 description 1
- VUGWHBXPMAHEGZ-SRVKXCTJSA-N Arg-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCN=C(N)N VUGWHBXPMAHEGZ-SRVKXCTJSA-N 0.000 description 1
- MLJZMGIXXMTEPO-UBHSHLNASA-N Asn-Trp-Ser Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CO)C(O)=O MLJZMGIXXMTEPO-UBHSHLNASA-N 0.000 description 1
- CSEJMKNZDCJYGJ-XHNCKOQMSA-N Asp-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)O)N)C(=O)O CSEJMKNZDCJYGJ-XHNCKOQMSA-N 0.000 description 1
- GIKOVDMXBAFXDF-NHCYSSNCSA-N Asp-Val-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O GIKOVDMXBAFXDF-NHCYSSNCSA-N 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- ZLHPWFSAUJEEAN-KBIXCLLPSA-N Cys-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CS)N ZLHPWFSAUJEEAN-KBIXCLLPSA-N 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 108091081406 G-quadruplex Proteins 0.000 description 1
- DTCCMDYODDPHBG-ACZMJKKPSA-N Gln-Ala-Cys Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CS)C(O)=O DTCCMDYODDPHBG-ACZMJKKPSA-N 0.000 description 1
- XQDGOJPVMSWZSO-SRVKXCTJSA-N Gln-Pro-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCC(=O)N)N XQDGOJPVMSWZSO-SRVKXCTJSA-N 0.000 description 1
- RUFHOVYUYSNDNY-ACZMJKKPSA-N Glu-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(O)=O RUFHOVYUYSNDNY-ACZMJKKPSA-N 0.000 description 1
- MXOODARRORARSU-ACZMJKKPSA-N Glu-Ala-Ser Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CCC(=O)O)N MXOODARRORARSU-ACZMJKKPSA-N 0.000 description 1
- JWNZHMSRZXXGTM-XKBZYTNZSA-N Glu-Ser-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JWNZHMSRZXXGTM-XKBZYTNZSA-N 0.000 description 1
- 108010053070 Glutathione Disulfide Proteins 0.000 description 1
- YYQGVXNKAXUTJU-YUMQZZPRSA-N Gly-Cys-His Chemical compound NCC(=O)N[C@@H](CS)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O YYQGVXNKAXUTJU-YUMQZZPRSA-N 0.000 description 1
- IXKRSKPKSLXIHN-YUMQZZPRSA-N Gly-Cys-Leu Chemical compound [H]NCC(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(O)=O IXKRSKPKSLXIHN-YUMQZZPRSA-N 0.000 description 1
- BMWFDYIYBAFROD-WPRPVWTQSA-N Gly-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN BMWFDYIYBAFROD-WPRPVWTQSA-N 0.000 description 1
- NDKSHNQINMRKHT-PEXQALLHSA-N His-Ile-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CC1=CN=CN1)N NDKSHNQINMRKHT-PEXQALLHSA-N 0.000 description 1
- MPXGJGBXCRQQJE-MXAVVETBSA-N His-Ile-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O MPXGJGBXCRQQJE-MXAVVETBSA-N 0.000 description 1
- CCUSLCQWVMWTIS-IXOXFDKPSA-N His-Thr-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O CCUSLCQWVMWTIS-IXOXFDKPSA-N 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 101710099623 Interferon lambda-1 Proteins 0.000 description 1
- 108010047761 Interferon-alpha Proteins 0.000 description 1
- 102000006992 Interferon-alpha Human genes 0.000 description 1
- 102000003996 Interferon-beta Human genes 0.000 description 1
- 108090000467 Interferon-beta Proteins 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- OHZIZVWQXJPBJS-IXOXFDKPSA-N Leu-His-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OHZIZVWQXJPBJS-IXOXFDKPSA-N 0.000 description 1
- IEWBEPKLKUXQBU-VOAKCMCISA-N Leu-Leu-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IEWBEPKLKUXQBU-VOAKCMCISA-N 0.000 description 1
- LZHJZLHSRGWBBE-IHRRRGAJSA-N Leu-Lys-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O LZHJZLHSRGWBBE-IHRRRGAJSA-N 0.000 description 1
- ZDJQVSIPFLMNOX-RHYQMDGZSA-N Leu-Thr-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N ZDJQVSIPFLMNOX-RHYQMDGZSA-N 0.000 description 1
- QWWPYKKLXWOITQ-VOAKCMCISA-N Leu-Thr-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(C)C QWWPYKKLXWOITQ-VOAKCMCISA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- XOQMURBBIXRRCR-SRVKXCTJSA-N Lys-Lys-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCCN XOQMURBBIXRRCR-SRVKXCTJSA-N 0.000 description 1
- DRRXXZBXDMLGFC-IHRRRGAJSA-N Lys-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCCN DRRXXZBXDMLGFC-IHRRRGAJSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 102100032341 PCNA-interacting partner Human genes 0.000 description 1
- 101710196737 PCNA-interacting partner Proteins 0.000 description 1
- 241000144300 Peromyscus gossypinus Species 0.000 description 1
- 229920002562 Polyethylene Glycol 3350 Polymers 0.000 description 1
- SKICPQLTOXGWGO-GARJFASQSA-N Pro-Gln-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCC(=O)N)C(=O)N2CCC[C@@H]2C(=O)O SKICPQLTOXGWGO-GARJFASQSA-N 0.000 description 1
- RMJZWERKFFNNNS-XGEHTFHBSA-N Pro-Thr-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O RMJZWERKFFNNNS-XGEHTFHBSA-N 0.000 description 1
- GZNYIXWOIUFLGO-ZJDVBMNYSA-N Pro-Thr-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GZNYIXWOIUFLGO-ZJDVBMNYSA-N 0.000 description 1
- ZMLRZBWCXPQADC-TUAOUCFPSA-N Pro-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 ZMLRZBWCXPQADC-TUAOUCFPSA-N 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- BTKUIVBNGBFTTP-WHFBIAKZSA-N Ser-Ala-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)NCC(O)=O BTKUIVBNGBFTTP-WHFBIAKZSA-N 0.000 description 1
- MOVJSUIKUNCVMG-ZLUOBGJFSA-N Ser-Cys-Ser Chemical compound C([C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(=O)O)N)O MOVJSUIKUNCVMG-ZLUOBGJFSA-N 0.000 description 1
- OJPHFSOMBZKQKQ-GUBZILKMSA-N Ser-Gln-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CO OJPHFSOMBZKQKQ-GUBZILKMSA-N 0.000 description 1
- XVWDJUROVRQKAE-KKUMJFAQSA-N Ser-Phe-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)CC1=CC=CC=C1 XVWDJUROVRQKAE-KKUMJFAQSA-N 0.000 description 1
- CKDXFSPMIDSMGV-GUBZILKMSA-N Ser-Pro-Val Chemical compound [H]N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O CKDXFSPMIDSMGV-GUBZILKMSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- MPUMPERGHHJGRP-WEDXCCLWSA-N Thr-Gly-Lys Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)O)N)O MPUMPERGHHJGRP-WEDXCCLWSA-N 0.000 description 1
- YUOCMLNTUZAGNF-KLHWPWHYSA-N Thr-His-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N2CCC[C@@H]2C(=O)O)N)O YUOCMLNTUZAGNF-KLHWPWHYSA-N 0.000 description 1
- XIULAFZYEKSGAJ-IXOXFDKPSA-N Thr-Leu-His Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CNC=N1 XIULAFZYEKSGAJ-IXOXFDKPSA-N 0.000 description 1
- IQPWNQRRAJHOKV-KATARQTJSA-N Thr-Ser-Lys Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCCN IQPWNQRRAJHOKV-KATARQTJSA-N 0.000 description 1
- RVMNUBQWPVOUKH-HEIBUPTGSA-N Thr-Ser-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O RVMNUBQWPVOUKH-HEIBUPTGSA-N 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- ASQFIHTXXMFENG-XPUUQOCRSA-N Val-Ala-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O ASQFIHTXXMFENG-XPUUQOCRSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 108010059722 Viral Fusion Proteins Proteins 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000001028 anti-proliverative effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 239000012911 assay medium Substances 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 210000000621 bronchi Anatomy 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 229910002091 carbon monoxide Inorganic materials 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 239000013522 chelant Substances 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000013024 dilution buffer Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000002845 discoloration Methods 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000007783 downstream signaling Effects 0.000 description 1
- 238000003255 drug test Methods 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 238000002270 exclusion chromatography Methods 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 229940125874 fusion protein inhibitor Drugs 0.000 description 1
- 235000004554 glutamine Nutrition 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- YPZRWBKMTBYPTK-BJDJZHNGSA-N glutathione disulfide Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@H](C(=O)NCC(O)=O)CSSC[C@@H](C(=O)NCC(O)=O)NC(=O)CC[C@H](N)C(O)=O YPZRWBKMTBYPTK-BJDJZHNGSA-N 0.000 description 1
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 230000010196 hermaphroditism Effects 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 238000010874 in vitro model Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000002664 inhalation therapy Methods 0.000 description 1
- 229960003130 interferon gamma Drugs 0.000 description 1
- 108010045648 interferon omega 1 Proteins 0.000 description 1
- 108700027921 interferon tau Proteins 0.000 description 1
- 229960001388 interferon-beta Drugs 0.000 description 1
- 108040006870 interleukin-10 receptor activity proteins Proteins 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 239000012669 liquid formulation Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 235000018977 lysine Nutrition 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- 239000003595 mist Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 235000008729 phenylalanine Nutrition 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 108010090894 prolylleucine Proteins 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 1
- 238000011552 rat model Methods 0.000 description 1
- 238000003030 reporter gene method Methods 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 229910021654 trace metal Inorganic materials 0.000 description 1
- 235000002374 tyrosine Nutrition 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
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
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/21—Interferons [IFN]
- A61K38/217—IFN-gamma
-
- 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
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/20—Interleukins [IL]
-
- 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
- A61K47/02—Inorganic compounds
-
- 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
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
本申请公开一种白细胞介素29突变体蛋白制剂,其特征在于,其包含:白细胞介素29突变体蛋白、缓冲体系、金属离子螯合剂和渗透压调节剂。本申请的IL‑29突变体蛋白制剂极大的减少了IL‑29突变体蛋白在储存过程中的降解和聚集,且IL‑29突变体蛋白制剂中的杂质增长速度缓慢,显著提高了IL‑29突变体蛋白稳定性。
Description
技术领域
本申请属于药物制剂领域。具体涉及一种白细胞介素29(IL-29)突变体蛋白制剂。
背景技术
干扰素是一类重要的家族性细胞因子,具有广谱的抗病毒和免疫调节作用。迄今,已经鉴定到7种(α、β、ω、δ、τ、γ、λ)形式的干扰素,它们分为三个大组:I型、II型和III型。所谓的“I型”干扰素包括干扰素α、干扰素β、干扰素ω、干扰素δ、干扰素τ。目前,干扰素γ是仅有的II型干扰素。III型干扰素是最近发现的一类细胞因子家族,包括干扰素λ1、λ2和λ3,也称为IL-28A、IL-28B和IL-29。
IL-28A、IL-28B和IL-29与I型干扰素有序列同源性,并与IL-10有基因序列的同源性。从功能上说,IL-28和IL-29类似于I型干扰素,均能诱导细胞的抗病毒作用,与I型干扰素不同的是,它们没有显示出对某些B细胞系的抗增殖活性。
野生型IL-29(干扰素λ1,简写IFN-λ1)基因编码200个氨基酸的蛋白,如SEQ IDNO:3所示。其中在该序列中1-19个氨基酸为信号肽序列,该蛋白成熟氨基酸序列为181个氨基酸,如SEQ ID NO:2所示。IL-29分子是由A-F六个蛋白质螺旋组成,其中螺旋A、C、D和F形成一个经典的up-up-down-down四螺旋束。IL-29通过与其受体复合物相互作用而启动下游信号通路,所述受体复合物由IFN-λRl和IL-10R2组成。值得注意的是,IFN-λRl是IFN-λ信号通路所特有。IFN-λ1与IFN-λRl特异性结合形成IFN-λ1/IFN-λRl复合物,其中IFN-λ1与IFN-λRl结合的活性中心的氨基酸残基分别为Pro25,Leu28,lys32,Arg35,Asp36,Glu39,Trp47,Phe152,Phe155,Arg156,Arg160。
无论是I型干扰素、II型干扰素还是III型干扰素,作为蛋白质药物,由于稳定性差、活性低,体内半衰期短等因素,在临床治疗使用中受到很大限制。因此,通过基因工程技术获得更为稳定、比活性更高的干扰素重组蛋白药物是人们所期望的。特别是利用雾化吸入疗法预防和/或治疗呼吸系统疾病时,需要雾化装置将药物溶液雾化成微小颗粒,吸入呼吸道及肺部使药物沉积在呼吸道和肺部,这种情况对于药物的稳定性和活性要求更高,因此人们更渴望获得稳定性更高且活性更好的IL-29突变体蛋白,并且亟需一种稳定的、可供临床使用的、适宜存储的IL-29突变体蛋白制剂。
发明内容
为了解决上述问题,本申请的目的在于提供一种稳定的并适合长期储存的IL-29突变体蛋白制剂。
本申请的具体技术方案如下:
1、一种白细胞介素29突变体蛋白制剂,其特征在于,其包含:白细胞介素29突变体蛋白、缓冲体系、金属离子螯合剂和渗透压调节剂。
2、根据项1所述的制剂,其特征在于,所述白细胞介素29突变体蛋白包含SEQ IDNO:1所示氨基酸序列上第161或第162位氨基酸的取代突变,其中所述第161位的天冬氨酸(D)或者第162位的甘氨酸(G)被其它天然氨基酸取代。
3、根据项1或2所述的制剂,其特征在于,所述第161位的天冬氨酸(D)被谷氨酸、苏氨酸或丝氨酸取代,或者第162位的甘氨酸(G)被脂肪族氨基酸取代。
4、根据项1~3中任一项所述的制剂,其特征在于,所述白细胞介素29突变体蛋白包含如下的氨基酸序列:SEQ ID NO:4,SEQ ID NO:6或SEQ ID NO:8。
5、根据项1~4中任一项所述的制剂,其特征在于,所述白细胞介素29突变体蛋白还包含SEQ ID NO:1所示氨基酸序列上第165位从半胱氨酸(C)到丝氨酸(S)的取代突变。
6、根据项1~5中任一项所述的制剂,其特征在于,所述白细胞介素29突变体蛋白包含如下的氨基酸序列:SEQ ID NO:5,SEQ ID NO:7或SEQ ID NO:9。
7、根据项1~6中任一项所述的制剂,其特征在于,所述白细胞介素29突变体蛋白的质量体积浓度为0.1~1mg/mL。
8、根据项1~7中任一项所述的制剂,其特征在于,所述缓冲体系为磷酸盐缓冲液、醋酸盐缓冲液或组氨酸盐缓冲液,优选为磷酸盐缓冲液。
9、根据项1~8中任一项所述的制剂,其特征在于,所述制剂的pH为4.0~5.0,优选为4.0~4.5。
10、根据项1~9中任一项所述的制剂,其特征在于,所述磷酸盐缓冲液的物质的量浓度为20~80mmol/L,优选为20~40mmol/L。
11、根据项1~10中任一项所述的制剂,其特征在于,所述金属离子螯合剂为依地酸二钠。
12、根据项1~11中任一项所述的制剂,其特征在于,所述依地酸二钠的质量体积浓度为0.01~0.1mg/mL,优选为0.01~0.05mg/mL。
13、根据项1~12中任一项所述的制剂,其特征在于,所述渗透压调节剂为氯化钠。
14、根据项1~13中任一项所述的制剂,其特征在于,所述氯化钠的质量体积浓度为6~10mg/mL。
15、根据项1~14中任一项所述的制剂,其特征在于,所述白细胞介素29突变体蛋白的质量体积浓度为0.5~1mg/mL,所述依地酸二钠的质量体积浓度为0.01mg/mL。
发明的效果
本申请的IL-29突变体蛋白制剂极大的减少了IL-29突变体蛋白在储存过程中的降解和聚集,且IL-29突变体蛋白制剂中的杂质增长速度缓慢,显著提高了IL-29突变体蛋白稳定性。
尤其是在使用磷酸盐缓冲液体系、且控制金属螯合剂的用量较低水平时,一方面保证了无刺激性,另一方面安全性更高,更适用于直达体内器官、对安全性要求高的剂型,如吸入剂型。
附图说明
图1和图2为实施例2的IL29突变体SDS-PAGE电泳图(自左至右,图1中,泳道1~4依次为IL29对照品、IL29DE、IL29DS、IL29GA;图2中,泳道1~4依次为IL29CS、IL29DE+CS、IL29DS+CS、IL29GA+CS)。
图3~图10分别为IL29对照品、IL29DE、IL29DS、IL29GA、IL29CS、IL29DE+CS、IL29DS+CS、IL29GA+CS的稳定性反相高效液相纯度图谱(其中主峰含量高的代表的是0点测定曲线,主峰含量低的代表为14天的曲线)。
图11为IL29突变体的雾化稳定性实验中气溶胶收集装置的图。
图12为利巴韦林和不同给药方案下的IL29突变体对RSV感染小鼠模型体内药效结果。
图13为利巴韦林和不同给药方案下的IL29突变体对RSV感染棉鼠模型体内药效结果。
具体实施方式
下面将更详细地描述本申请的具体实施方式。需要说明的是,在通篇说明书及权利要求当中所提及的“包含”或“包括”为一开放式用语,故应解释成“包含但不限定于”。说明书后续描述为实施本申请的较佳实施方式,然所述描述乃以说明书的一般原则为目的,并非用以限定本申请的范围。本申请的保护范围当视所附权利要求所界定者为准。
申请相关术语
本申请中的“突变蛋白”指野生型蛋白氨基酸序列发生改变,例如通过基因工程方法使野生型蛋白氨基酸序列突变获得的蛋白。在本申请中“突变蛋白”、“突变体蛋白”或“突变体”表达相同含义,可互换使用。
本申请中的“依地酸二钠”为乙二胺四乙酸二钠,为无味无臭或微咸的白色或乳白色结晶或颗粒状粉末,无臭、无味。它能溶于水,极难溶于乙醇。它是一种重要的螯合剂,能螯合溶液中的金属离子。防止金属引起的变色、变质、变浊和维生素C的氧化损失,还能提高油脂的抗氧化性(油脂中的微量金属如铁、铜等有促进油脂氧化的作用)。化学式为C10H14N2Na2O8,它有六个配位原子,形成的配合物叫做螯合物,依地酸二钠在配位滴定中经常用到,一般是测定金属离子的含量。其在染料、食品、药品等工业上有重要用途。本申请中的“依地酸二钠二水合物”又称乙二胺四乙酸二钠盐二水合物,分子式为C10H14N2Na2O8·2H2O,其为白色结晶粉末,无臭,应用领域覆盖化工、医药、食品、农业等多个行业,具有良好的络合效应。
本申请提供一种白细胞介素29突变体蛋白制剂,其中,其包含:白细胞介素29突变体蛋白、缓冲体系、金属离子螯合剂和渗透压调节剂。
在一个具体实施方式中,本申请的制剂中,所述IL-29突变体蛋白包含SEQ ID NO:1所示氨基酸序列上第161或第162位氨基酸的取代突变,其中所述第161位的天冬氨酸(D)或者第162位的甘氨酸(G)被其它天然氨基酸取代,例如被选自如下的氨基酸取代:甘氨酸、丙氨酸、缬氨酸、亮氨酸、异亮氨酸、甲硫氨酸(蛋氨酸)、脯氨酸、色氨酸、丝氨酸、酪氨酸、半胱氨酸、苯丙氨酸、天冬酰胺、谷氨酰胺、苏氨酸、天冬氨酸、谷氨酸、赖氨酸、精氨酸或组氨酸。
本领域技术人员可以理解,SEQ ID NO:1的第161位和第162位与SEQ ID NO:2(野生型白细胞介素29成熟蛋白)和SEQ ID NO:3(包含信号肽的野生型白细胞介素29全长蛋白)的相应位置的对应关系,因此,基于SEQ ID NO:2或SEQ ID NO:3(或来源于SEQ ID NO:2或SEQ ID NO:3的不同长度的氨基酸序列)相应位置的氨基酸突变,也涵盖在本申请的保护范围内。本申请的保护范围还涵盖来源于SEQ ID NO:2或SEQ ID NO:3、但具有不同长度的氨基酸序列的上述第161位和第162位对应位置包含取代突变的白细胞介素29突变体蛋白。
在一个具体实施方式中,本申请的制剂中,所述IL-29突变体蛋白包含SEQ ID NO:1所示氨基酸序列上第161或第162位氨基酸的取代突变,其中所述第161位的天冬氨酸(D)或者第162位的甘氨酸(G)被其它天然氨基酸取代,还包含起始蛋氨酸(M)。这是因为IL-29在原核细胞(例如E.coli)中表达时,则表达的IL-29蛋白存在N-末端或氨基末端的甲硫氨酸。
在一个具体实施方式中,本申请的制剂中,所述IL-29突变体蛋白包含SEQ ID NO:1所示氨基酸序列上第161或第162位氨基酸的取代突变,即从SEQ ID NO:1所示的蛋白的N-末端或者氨基-末端算起,在第161或第162位上的突变,例如所述第161位的天冬氨酸(D)或者第162位的甘氨酸(G)被其它天然氨基酸取代。在一个具体实施方式中,本申请的IL-29突变体蛋白是在原核细胞(例如E.coli)中表达的突变体蛋白,包含在第162或第163位(因为加了N-末端M,从M算起)上的取代突变,例如所述第162位的天冬氨酸(D)或者第163位的甘氨酸(G)被其它天然氨基酸取代。在一个具体实施方式中,所述IL-29突变体蛋白如SEQ IDNO:4-9所示。
在一个具体实施方式中,本申请的制剂中,所述IL-29突变体蛋白包含SEQ ID NO:1所示氨基酸序列上第161或第162位氨基酸的取代突变,例如其中所述第161位的天冬氨酸(D)被谷氨酸、苏氨酸或丝氨酸取代,或者第162位的甘氨酸(G)被脂肪族氨基酸取代;SEQID NO:1的氨基酸序列对应的基因序列为SEQ ID NO:18。在一个具体实施方式中,所述IL-29突变体蛋白是在原核细胞(例如E.coli)中表达的突变体蛋白,包含在第162或第163位(因为加了N-末端M,从M算起)上的取代突变,例如其中所述第162位的天冬氨酸(D)被谷氨酸、苏氨酸或丝氨酸取代,或者第163位的甘氨酸(G)被脂肪族氨基酸取代。
在一个具体实施方式中,本申请的制剂中,所述IL-29突变体蛋白包含如下的氨基酸序列或由如下的氨基酸序列组成:SEQ ID NO:4,SEQ ID NO:6或SEQ ID NO:8。
在一个具体实施方式中,本申请的制剂中,所述IL-29突变体蛋白还包含SEQ IDNO:1所示氨基酸序列上第165位从半胱氨酸(C)到丝氨酸(S)的取代突变。同上所述,当IL-29突变体蛋白在原核细胞(例如E.coli)中表达时,所述第165位从半胱氨酸(C)到丝氨酸(S)的取代突变则会出现在第166位,此时,所述IL-29突变体蛋白序列如SEQ ID NO:10所示。
在一个具体实施方式中,本申请的制剂中,所述IL-29突变体蛋白包含如下的氨基酸序列或由如下的氨基酸序列组成:SEQ ID NO:5,SEQ ID NO:7或SEQ ID NO:9。
在一个具体实施方式中,本申请的制剂中,所述IL-29突变体蛋白为如以下SEQ IDNO:4所示的IL-29突变体蛋白(IL-29DE):
MKPTTTGKGCHIGRFKSLSPQELASFKKARDALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAELALTLKVLEAAAGPALEDVLDQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQEAPKKESAGCLEASVTFNLFRLLTRDLKYV AEGNLCLRTS THPEST(SEQ ID NO:4)
在一个具体实施方式中,本申请的制剂中,所述IL-29突变体蛋白为如以下SEQ IDNO:5所示的IL-29突变体蛋白(IL-29DE+CS):
MKPTTTGKGCHIGRFKSLSPQELASFKKARDALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAELALTLKVLEAAAGPALEDVLDQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQEAPKKESAGCLEASVTFNLFRLLTRDLKYV AEGNLSLRTS THPEST(SEQ ID NO:5)
在一个具体实施方式中,本申请的制剂中,所述IL-29突变体蛋白为如以下SEQ IDNO:6所示的IL-29突变体蛋白(IL-29DS):
MKPTTTGKGCHIGRFKSLSPQELASFKKARDALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAELALTLKVLEAAAGPALEDVLDQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQEAPKKESAGCLEASVTFNLFRLLTRDLKYV ASGNLCLRTS THPEST(SEQ ID NO:6)
在一个具体实施方式中,本申请的制剂中,所述IL-29突变体蛋白为如以下SEQ IDNO:7所示的IL-29突变体蛋白(IL-29DS+CS):
MKPTTTGKGCHIGRFKSLSPQELASFKKARDALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAELALTLKVLEAAAGPALEDVLDQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQEAPKKESAGCLEASVTFNLFRLLTRDLKYV ASGNLSLRTSTHPEST(SEQ ID NO:7)
在一个具体实施方式中,本申请的制剂中,所述IL-29突变体蛋白为如以下SEQ IDNO:8所示的IL-29突变体蛋白(IL-29GA):
MKPTTTGKGC HIGRFKSLSP QELASFKKAR DALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAE LALTLKVLEAAAGPALEDVL DQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQ EAPKKESAGC LEASVTFNLFRLLTRDLKYV ADANLCLRTSTHPEST(SEQ ID NO:8)
在一个具体实施方式中,本申请的制剂中,所述IL-29突变体蛋白为如SEQ ID NO:9所示的IL-29突变体蛋白(IL-29GA+CS):
MKPTTTGKGCHIGRFKSLSPQELASFKKARDALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAELALTLKVLEAAAGPALEDVLDQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQEAPKKESAGCLEASVTFNLFRLLTRDLKYVADANLSLRTS THPEST(SEQ ID NO:9)
在一个具体实施方式中,本申请的制剂中,所述IL-29突变体蛋白还包含为促进蛋白纯化的短序列(例如6个组氨酸的短序列),或者为延长半衰期的短氨基酸序列。
在一个具体实施方式中,本申请的制剂为液体制剂,其可以制成针剂、片剂、胶囊、吸入剂、栓剂等剂型。优选地,本申请的制剂可以制成吸入剂,例如干粉吸入剂或液体吸入剂、雾化吸入剂、气雾剂、柔雾剂和喷雾剂等,通过吸入装置,例如雾化吸入装置、定量吸入装置、干粉吸入装置给药。
在一个具体实施方式中,本申请的制剂中,所述白细胞介素29突变体蛋白的质量体积浓度为0.1~1mg/mL,例如可为0.1mg/mL、0.2mg/mL、0.3mg/mL、0.4mg/mL、0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL、1mg/mL等。
在一个具体实施方式中,本申请的制剂中,所述缓冲体系为磷酸盐缓冲液、醋酸盐缓冲液或组氨酸盐缓冲液,优选为磷酸盐缓冲液。其中,所述缓冲液的物质的量浓度为20~80mmol/L,优选为20~40mmol/L。因磷酸盐缓冲液无刺激性,且在多种已上市产品中使用,安全性更好,故优选磷酸盐缓冲液;所述磷酸盐缓冲液的物质的量浓度为20~80mmol/L,例如可为20mmol/L、25mmol/L、30mmol/L、35mmol/L、40mmol/L、45mmol/L、50mmol/L、55mmol/L、60mmol/L、65mmol/L、70mmol/L、75mmol/L、80mmol/L等,优选为20~40mmol/L。
在一个具体实施方式中,所述制剂的pH为4.0~5.0,例如可为4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9,5.0等,优选为4.0~4.5。
在一个具体实施方式中,本申请的制剂中,所述金属离子螯合剂为依地酸二钠二水合物,其在所述制剂中的质量体积浓度为0.01~0.1mg/mL,例如可为0.01mg/mL、0.02mg/mL、0.03mg/mL、0.04mg/mL、0.05mg/mL、0.06mg/mL、0.07mg/mL、0.08mg/mL、0.09mg/mL、0.1mg/mL等,优选为0.01~0.05mg/mL。所述金属离子螯合剂可降低由金属离子引起的蛋白降解反应。
在一个具体实施方式中,本申请的制剂中,所述渗透压调节剂的含量使所述制剂的渗透压保持在280~320mOsmol/kg。其中,所述渗透压调节剂选自氯化钠、氯化钾和氯化镁中的一种或几种。优选地,所述渗透压调节剂为氯化钠,所述氯化钠的质量体积浓度为6~10mg/mL,例如可为6mg/mL、6.5mg/mL、7mg/mL、7.5mg/mL、8mg/mL、8.5mg/mL、9mg/mL、9.5mg/mL、10mg/mL等。渗透压调节剂可以使制剂与人体内各液体的渗透压保持平衡,维持等渗。
在一个具体实施方式中,本申请的制剂中,所述白细胞介素29突变体蛋白的质量体积浓度为0.5~1mg/mL,所述依地酸二钠的质量体积浓度为0.01mg/mL,所述制剂的pH为4.0~5.0。
在一个具体实施方式中,本申请的制剂中,所述制剂为:0.5mg/mL白细胞介素29突变体蛋白,0.01mg/mL依地酸二钠,20mmol/L磷酸盐缓冲液、8mg/mL氯化钠,所述制剂的pH为4.5。
在一个具体实施方式中,本申请的制剂中,所述制剂为:1.0mg/mL白细胞介素29突变体蛋白,0.01mg/mL依地酸二钠,20mmol/L磷酸盐缓冲液、8mg/mL氯化钠,所述制剂的pH为4.5。
在一个具体实施方式中,本申请的制剂中,所述制剂为:0.5mg/mL白细胞介素29突变体蛋白,0.05mg/mL依地酸二钠,20mmol/L磷酸盐缓冲液、8mg/mL氯化钠,所述制剂的pH为4.5。
在一个具体实施方式中,本申请的制剂中,所述制剂为:1.0mg/mL白细胞介素29突变体蛋白,0.05mg/mL依地酸二钠,20mmol/L磷酸盐缓冲液、8mg/mL氯化钠,所述制剂的pH为4.5。
白细胞介素29(IL-29)突变体蛋白和IL-29突变体蛋白制剂的稳定性的检测方法也包括很多种,如反相高效液相色谱可以用来分析疏水性和极性有差异的杂质;离子交换高效液相色谱可以用来分离电荷差异较大的杂质;而分子筛排阻色谱用来分析二聚体、高聚体和单体。每种检测方法关注点不同,但均可用来表征蛋白纯度得知该蛋白制剂的稳定性。
以下利用实施例对本申请做以详细说明。然而应当理解,可以以各种形式实现本申请而不应被这里阐述的实施例所限制。相反,提供这些实施例是为了能够更透彻地理解本发明,并且能够将本申请的范围完整的传达给本领域的技术人员。
实施例
实施例1IL-29突变体蛋白的制备
1.1突变体蛋白表达工程菌的构建
使用化学合成的方式获得IL-29突变体蛋白基因片段SEQ ID NO:11-17,通过NodeI和Xho I位点,将上述片段插入原核表达质粒pET-30a(+)(Novagen)中并测序验证。得到的用于转化测定的表达质粒。将上述获得的含有目的基因的质粒转化大肠杆菌BL21(DE3)感受态细胞(Invitrogen),将BL21感受态细胞50μL置于冰浴上融化,加入质粒,轻轻摇匀,并在冰浴中放置30分钟。继而42℃水浴热激30秒,然后快速将离心管转移到冰浴中,放置2分钟,该过程不要摇动离心管。向离心管中加入500μL无菌的LB培养基(不含抗生素),混匀后置于37℃,180rpm培养1小时,使细菌复苏。吸取200μL已转化的感受态细胞加到含有卡那霉素抗性的LB琼脂培养基平板上,将细胞均匀涂开。将平板置于37℃至液体被吸收,倒置平板,37℃过夜培养。次日,使用接种环挑取转化平皿中的单克隆菌落,并接种于15mL的无菌LB培养基(含卡那霉素),30℃过夜培养。
1.2 IL-29突变体蛋白的表达和纯化
向50mL的LB培养基中加入50μL上述细菌的菌液,同时加入50μL卡那霉素,混匀后放30℃恒温振荡器中,接种过夜。取过夜接种的菌液10mL加入1000mL的LB培养基中,同时加入1000μL卡那霉素。摇匀后放于37℃摇床内,200rpm,培养至菌液OD600为0.4-0.6小时,加入终浓度为0.5mM IPTG诱导,继续培养4h后收集菌体。表达的IL-29突变体约占菌体总蛋白的30-50%,主要以包涵体形式存在。
将发酵菌体以TE(10mmol/L Tris-HCl,1mmol/L EDTA,pH 6.5)溶液(m:V=1:10)洗涤菌体3次,然后60Mpa高压匀浆破碎,匀浆后,镜检破菌率。当菌体破碎率约95%时(约破菌2~3次),8000rpm离心15min,收集破碎菌体沉淀。取破碎菌体沉淀置于烧杯中,加入包涵体洗涤液(10mM Tris-HCl+1mM EDTA+0.5%Triton-X100,pH 6.5,m:V=1:10),于磁力搅拌机上搅拌30min,洗涤3~5次。包涵体用包涵体裂解液(7M盐酸胍+50mM Tris-HCl+10mMDTT,pH 6.5,m:V=1:10)裂解,室温搅拌,过夜。裂解的蛋白慢速加入复性液(100mM Tris-HCl,0.5M精氨酸,0.5%PEG3350(m:V),2mM GSH:0.5mM GSSG,pH 8.5)中,使蛋白终浓度为0.2mg/mL,室温搅拌,过夜。
复性液8000rpm离心5min,收集上清。利用超滤膜包,超滤膜孔径为10kDa,用20mM磷酸盐缓冲液pH 7.0平衡超滤膜,然后将1L上清液浓缩10倍。浓缩液加入5倍体积的注射用水稀释待上样,Sepharose FF填料装柱,50mmol/L Tris-HCl,pH8.5,0.1mol/L NaCl平衡,上样;然后用50mmol/L Tris-HCl,pH8.5,0.15mol/L NaCl进行洗脱,收集洗脱峰组分最终收集样品溶液600mL;Sepharose FF填料装柱,20mmol/L磷酸盐,pH7.4,0.05mol/L NaCl平衡,上样,直到检测器基线平稳。用20mmol/L磷酸盐,pH7.4,0.2mol/L NaCl冲洗,收集洗脱峰组分。
得到七种IL-29突变体蛋白(简称IL-29突变体),分别对应于蛋白氨基酸序列SEQID NO:4-10,分别命名为IL-29DE、IL-29DE+CS、IL-29DS、IL-29DS+CS、IL-29GA、IL-29GA+CS、IL-29CS。
其中,IL29 DE+CS的基因序列如SEQ ID NO:11所示,L29 DS+CS的基因序列如SEQID NO:12所示,IL29 GA+CS的基因序列如SEQ ID NO:13所示,IL29 DE的基因序列如SEQ IDNO:14所示,IL29 DS的基因序列如SEQ ID NO:15所示,IL29 GA的基因序列如SEQ ID NO:16所示,IL29CS的基因序列如SEQ ID NO:17所示。
同时,用上述同样的方法也合成了IL-29野生型蛋白(简称IL-29对照品),对应于蛋白氨基酸序列SEQ ID NO:1并且N末端有额外的M氨基酸。
实施例2检测获得的IL-29突变体的各种指标
2.1 SDS-PAGE电泳检测获得IL-29突变体的分子量及纯度
利用SDS-PAGE电泳上样缓冲液,在加入巯基乙醇的情况下,将Marker及10μg的上述得到的蛋白分别上样,进行电泳,电泳条件为200V恒电压,45分钟。用考马斯亮蓝G-25进行染色,检测蛋白分子量及纯度,结果见图1和2所示。
由图1和2可知,IL29突变体蛋白及对照品的分子量分别为20kDa,说明所获得的目的蛋白正确,只有一条带无其他杂带,纯度可达到100%。
2.2采用色谱法检测IL-29突变体的体外反相高效液相纯度
2.2.1反相高效液相色谱法
配制流动相A:乙腈:水:三氟乙酸体积比为20:80:0.1,流动相B:乙腈:水:异丙醇:三氟乙酸体积比为70:20:10:0.1,使用色谱柱(XBridge BEH C18 Column,130A,5μm,4.6mm*100mm),分析各蛋白供试品。其中分析条件为:流速1.0mL/min,采集时间:65min,采集波长214nm,柱温45℃,洗脱梯度如下表:
表1
时间(min) | 0 | 5 | 5.01 | 45 | 55 | 55.01 | 65 |
B% | 27 | 27 | 27 | 49 | 70 | 27 | 27 |
平行进样两针供试品原液,根据样品浓度设定进样体积,进样量为15μg~20μg;最后,按照面积归一化法积分,计算供试品主峰纯度。
2.2.2离子交换色谱法
配制流动相A:25mmol/L磷酸盐缓冲液pH7.0,流动相B:25mmol/L磷酸盐缓冲液,0.5mol/L氯化钠pH6.7),使用色谱柱(Thermo ProPac WCX-10 4.0*250mm),分析各蛋白供试品。其中分析条件为流速0.8mL/min,采集时间:55min,采集波长214nm,柱温25℃,洗脱梯度:
表2
时间(min) | 0 | 30 | 31 | 40 | 41 | 55 |
A% | 80 | 70 | 10 | 10 | 80 | 80 |
B% | 200 | 30 | 90 | 90 | 20 | 20 |
进样量为20μg,按照面积归一化法积分,计算供试品平行两针的主峰纯度。
2.3采用报告基因法测定IL-29突变体的体外细胞生物学活性
使HEK293-ISRE-Luc细胞(购自中国食品药品检定研究院)在完全培养液中贴壁生长。按1:4传代,每周2~3次,于完全培养液中生长。取培养的细胞弃去培养液,用PBS洗涤1次后消化和收集细胞,用测定培养液(BIBCO)配制成每1mL含3.5×105~4.5×105个细胞的细胞悬液。将配制完成的IL-29突变体蛋白和IL-29对照品移入可用于细胞培养和化学发光酶标仪(MolecularDevices)读数的96孔板中,每孔加入100μL,然后将上述细胞悬液接种于同一96孔板中,每孔100μL。于37℃、5%二氧化碳条件下培养19~23小时。小心吸净96孔板中的上清液,按荧光素酶检测试剂盒(Bright-GloTM Luciferase Assay System,Promega)说明书加入细胞裂解液和荧光素酶底物,用化学发光酶标仪进行测定,分别记录IL-29突变体和IL-29对照品的EC50值。如下计算其相对生物学活性:以IL-29对照品作为标准,其0点活性定义为“100%”,相对生物学活性=IL-29对照品EC50(0点)/IL-29突变体EC50。
测定IL-29突变体各供试品的EC50,并将IL-29对照品作为对照品计算各突变体的相对生物学活性测定,具体结果如下表3:
表3 IL-29突变体的体外细胞生物学活性数据
蛋白类型 | EC<sub>50</sub>(ng/mL) | 相对生物学活性(%) |
IL-29对照品 | 4.783 | 100% |
IL-29DE | 1.697 | 282% |
IL-29DS | 4.316 | 90% |
IL-29GA | 5.264 | 91% |
IL-29CS | 4.639 | 103% |
IL-29DE+CS | 1.428 | 335% |
IL-29DS+CS | 4.982 | 96% |
IL-29GA+CS | 3.985 | 120% |
由上表3可知,虽然IL-29的第162位的D不位于与IL-29受体结合的活性中心,经实验意想不到的发现,单突变的IL-29DE的EC50仅仅为1.697,远远低于IL-29对照品的4.783,生物学活性竟然提高了近三倍,而其它的单突变位点的突变体IL-29DS、IL-29GA、IL-29CS与IL-29对照品相比EC50生物学活性基本无变化。双突变的IL-29DE+CS在IL-29的第162位的D突变的基础上再加上第166位的C突变为S,其生物活性在单突变的IL-29DE的基础上又有进一步的提高,但双突变的IL-29DS+CS、IL-29GA+CS突变体生物学活性与IL-29对照品相比均无明显提高。本结果说明,不位于与IL-29受体结合的活性中心第162位的D突变为E有想不到的提高突变后蛋白生物学活性的效果。
实施例3IL-29突变体的50℃稳定性检测结果
本申请中的蛋白的稳定性主要是由反相高效液相纯度表征的。
在50℃±2℃/75%相对湿度±5%相对湿度的条件下,按照表4取样,以与实施例2中2.2.1相同的方法进行IL-29突变体各供试品的反相高效液相纯度测定,同以与实施例2中2.3相同的方法进行IL-29突变体各供试品的生物学活性测定,结果详见表5。同时将表5中的0天和14天的纯度数值做对比色谱图,具体见图3~10。
表4 IL-29突变体在50℃的稳定性验证方案
表5 IL-29突变体在50℃时的反相高效液相纯度的测定结果
从上表5和图3~10结果可知,纯度降幅最大的两个供试品分别是IL-29对照品和IL-29CS,其中IL-29对照品在14天内纯度从85.82%急剧降到了34.46%,降幅高达51.36%,IL-29CS在14天内纯度从97.03%急剧降到了46.20%,降幅高达50.83%;降幅最小的两个供试品分别是IL-29DE和IL-29DE+CS,其中IL-29DE在14天内纯度从92.68%降到了77.39%,降幅仅有15.29%,IL-29DE+CS在14天内纯度从97.22%降到了86.66%,降幅更低只有10.56%。其它单点突变体IL-29DS、IL-29GA14天内的纯度降幅分别为24.22%和19.65%,其它双点突变体IL-29DS+CS、IL-29GA+CS14天内的纯度降幅分别为18.25%和14.97%。
从上述结果可推知,第162位位点从D突变为E的单点突变体中IL-29DE稳定性最好,在14天内纯度降幅最小。并且,因IL-29CS的稳定性远小于IL-29DE,按照常规推论,双点突变体IL-29DE+CS的稳定性可能会低于IL-29DE,而本发明中IL-29DE+CS的稳定性还远高于IL-29DE,说明此双点同时突变后在提高突变体稳定性方面还具有协同作用。同时检测突变体生物学活性,发现14天内突变体IL-29DE和IL-29DE+CS的生物学活性基本无变化。
实施例4 IL-29突变体的25℃光照稳定性检测结果
在25℃±2℃/60%相对湿度±5%相对湿度/5000±500勒克斯的条件下,按照如表6取样,以与实施例2中相同的方法进行IL-29突变体的稳定性测定,结果见表7,其中以IL-29CS作为对照品。
表6 IL-29突变体25℃光照稳定性方案
表7 IL-29突变体25℃光照时稳定性的测定结果
从以上表7的结果可以看出,IL-29突变体IL-29DE+CS的稳定性在25℃光照条件下的稳定性要好于IL-29CS。
实施例5 IL29各突变体的雾化稳定性实验
采用德国PARI LCD型的射流雾化器和TurboBOY N雾化泵进行雾化实验,共雾化2mL样品,并采用如下图11方式进行气溶胶收集,收集的样品以与实施例2中2.2.1相同的方法进行IL29突变体各供试品的反相高效液相纯度测定以验证各突变体的雾化稳定性。其中以IL29 CS作为对照品,结果见下表8。
表8
从上表8结果可知,IL29突变体IL29 DE+CS、IL29 GA+CS雾化后纯度仅下降3%左右,IL29 DS+CS雾化后纯度下降了8.1%,而IL29CS的雾化后纯度大大下降,竟然下降了近11%之多。由此可看出,IL29突变体的雾化稳定性与IL29CS相比显著增强,尤其是IL29突变体IL29 DE+CS、IL29 GA+CS雾化后稳定性特别好,特别适用于制备雾化吸入制剂。
实施例6 IL29突变体对RSV感染人支气管上皮细胞(HBEC)的体外药效测定
HBEC(human bronchial epithelial cell人支气管上皮细胞)的细胞分化(CellApplication)完成后,将倍比稀释的IL29突变体(IL29 DE+CS)、阳性对照药(BMS-433771,上海药明康德提供,为呼吸道合胞病毒融合蛋白抑制剂)加入分化后的HBEC细胞,并于37℃和5%CO2培养箱中孵育。呼吸道合胞病毒(respiratory syncytial virus,上海药明康德提供)在感染前24h,以及感染后1h和24h接种,活性测试孔中每孔加入滴度100TCID50(半数组织培养感染剂量)的RSV,细胞于37℃和5%CO2培养箱中培养3天后,应用RNA提取试剂盒(货号74181,Qiagen)提取细胞中RSV RNA,并用RT-qPCR定量。用GraphPad Prism软件分析化合物剂量反应曲线和计算EC50值及平均抑菌率,结果见表9及表10。
表9阳性对照药BMS-433771在HBEC细胞模型中对RSV感染的抑制
表10 IL29突变体在HBEC细胞模型中对RSV感染的抑制
结果表明,IL29 DE+CS在人支气管上皮细胞的体外模型中显示出很好的抗RSV药效,EC50为0.13ng/ml(6.5pM),是对照药的1/100000,远低于对照药。
实施例7 IL29突变体对RSV感染小鼠模型的体内药效测定
在第0天,所有小鼠(雌性,6-7周龄,16-18g,无特定病原体(SPF)级BALB/c小鼠(上海药明康德提供))通过腹腔注射戊巴比妥钠(75mg/kg)麻醉后,经滴鼻的方式接种RSV(人呼吸道合胞病毒A2(RSV-A2;,购自BEI Resources,NIAID,NIHBethesda,MD),接种量为1.1×105PFU每只动物,接种体积为50μL。
从第0天至第3天,根据表11方案对动物进行给药,给药方法为肺部喷雾小鼠经麻醉后,将预装药物溶液的液体雾化装置(购买于上海玉研仪器有限公司)的微喷头插管轻轻插入小鼠气管的合适位置,快速按压雾化器高压推送装置的活塞将定量体积的药物雾化至小鼠肺部(参考文献Joseph D.Brain,Dwyn E.Knudson,Sergei P.Sorokin,MichaelA.Davis,Pulmonary distribution of particles given by intratrachealinstillation or by aerosol inhalation,Environmental research 11,Volume 11,Issue 1,1976,Pages 13-33的给药方案),频率为每天一次。感染后第5天为体内实验终点,对所有动物进行安乐死并收取肺组织,检测其中的病毒滴度(空斑实验)。结果见表12及图12。
表11小鼠体内给药方案
注:本实施例中使用的IL29突变体为IL29 DE+CS。
表12不同药物对RSV感染小鼠模型体内药效结果
注:与溶媒(生理盐水)组相比,***P<0.001,**P<0.01
数据表明,RSV接种后可以在小鼠体内大量复制,阳性对照药物利巴韦林显著抑制了RSV在小鼠体内的复制,显示出预期的体内抗RSV活性证明此模型系统的有效性。利巴韦林的给药时间点为病毒感染前1h给药(预防模型),而受试药物IL29突变体在设定测定条件下(病毒感染前1小时、病毒感染后1小时和病毒感染后24小时),均可以显著抑制RSV病毒在小鼠体内的复制,其中接种前1小时(10μg)及接种后1小时(10μg)首次给药组小鼠肺组织中的病毒滴度均在检测下限以下,显示极佳的体内抗RSV药效。感染后24小时给药组也显示出了良好的抗RSV效果。利巴韦林在感染前1小时给药的预防模型结果相比,IL29突变体感染后1小时给药的治疗模型的抗病毒效果与其相当。这也充分说明了IL29突变体有着非常好的抗病毒治疗潜力。
实施例8 IL29突变体对RSV感染棉鼠模型的体内药效测定
在第0天,所有小鼠(Sigmodon hispidus棉鼠,雌雄各半,5周龄,SPF级别(购自Envigo))通过腹腔注射戊巴比妥钠(75mg/kg)麻醉后,经滴鼻的方式接种RSV(人呼吸道合胞病毒A2(RSV-A2),购自BEI Resources,NIAID,NIHBethesda,MD),接种量为1.1×105PFU每只动物,接种体积为50μL。
从第0天至第3天,根据表13测定方案对动物进行给药,给药方法为肺部喷雾(参考文献Joseph D.Brain,Dwyn E.Knudson,Sergei P.Sorokin,Michael A.Davis,Pulmonarydistribution of particles given by intratracheal instillation or by aerosolinhalation,Environmental research 11,Volume 11,Issue 1,1976,Pages 13-33的给药方案),频率为每天一次。在气管中插入一个22G针头的3ml注射器,向肺中注入2mL 0.9%生理盐水,收集支气管肺泡灌洗液(BALF)。收集的BALF分装于无菌1.5mLEP管中,置于干冰上冷冻,-80℃保存至空斑试验分析。结果见表14和图13。
表13棉鼠体内给药方案
注:本实施例中使用的IL29突变体为IL29 DE+CS。
表14不同药物对RSV感染棉鼠模型体内药效结果
表14和图13数据表明,RSV接种后可以在棉鼠体内大量复制,受试药物IL29突变体在设定条件下(接种后1小时给药的治疗模型),可以显著抑制RSV病毒在小鼠体内的复制,接种后1小时(1μg)首次给药组(第2、3、4组)棉鼠支气管灌洗液中的病毒滴度和对照组相比均有统计学差异,并且3个剂量组(第2-4组)显示出了剂量相关性,显示极佳的体内抗RSV药效。棉鼠被呼吸道合胞病毒感染后,病毒会在其肺部的支气管大量复制,而支气管灌洗液的病毒载量非常好的反映了的肺内的病毒复制情况。因此,IL29突变体显示除了非常好的对呼吸道合胞病毒的治疗潜力。
实施例9缓冲液pH筛选
称量醋酸钠固体3.4g,溶于800mL超纯水中,加稀盐酸/NaOH将pH值分别调pH为3.5、4.0、4.5、5.0、5.5、6.0,并分别定容至1L得到25mmol/L醋酸盐缓冲液。然后用已配制的稀释缓冲液将实施例1中的IL-29突变体蛋白IL-29DE+CS进行透析处理,透析置换缓冲液后将样品使用缓冲液稀配至浓度为100μg/mL,分装0.5mL/支样品。样品信息如下表15,使用与实施例2中2.2.1相同的方法进行反相高效液相纯度测定,使用与实施例2中2.2.2相同的方法进行离子交换纯度测定,同时使用反相高效液相色谱法进行蛋白定量。
表15
缓冲液pH筛选加速稳定性条件和取样设计如下表16所示:
表16
50℃加速实验反相高效液相纯度检测结果如下表17所示:
表17
50℃加速实验离子交换纯度检测结果如下表18所示:
表18
50℃加速实验反相高效液相色谱和离子交换纯度检测数据均显示,缓冲液pH值在4.0~5.0呈现较稳定状态;同时,50℃加速实验蛋白定量检测结果显示缓冲液pH值在3.5~6.0时蛋白定量检测均未出现明显的含量变化。优选缓冲液pH值在4.0~5.0之间,更优选缓冲液pH值在4.0~4.5之间。综合pH范围的数值结果,将该制剂最优选的pH值确定为4.5。这样,在大规模生产过程中,才可以将制剂控制在一个相对较优的pH值范围内。
实施例10缓冲液种类筛选
如下表19所示配制醋酸盐缓冲液、柠檬酸盐缓冲液、组氨酸缓冲液和磷酸盐缓冲液。
表19
用已配制的四种缓冲液分别将实施例1中的IL-29突变体蛋白IL-29DE+CS样品进行透析处理,透析置换为缓冲液后将样品使用缓冲液稀配至浓度为200μg/mL,分装0.5mL/支。样品信息如下表20,使用与实施例2中2.2.1相同的方法进行反相高效液相纯度测定,使用与实施例2中2.2.2相同的方法进行离子交换纯度测定,同时使用反相高效液相色谱法进行蛋白定量。
表20
缓冲液种类筛选加速稳定性条件及取样设计如下表21所示:
表21
40℃加速实验反相高效液相纯度检测结果如下表22所示:
表22
40℃加速实验离子交换纯度检测结果如下表23所示:
表23
25℃加速实验反相高效液相纯度检测结果如下表24所示:
表24
25℃加速实验离子交换纯度检测结果如下表25所示:
表25
40℃加速实验反相高效液相纯度检测数据显示,使用柠檬酸缓冲液处理的样品呈现较差的稳定性,使用其它三种缓冲液处理的样品均呈现较好的稳定性且无明显差异;40℃加速实验离子交换纯度检测数据显示,使用柠檬酸缓冲液处理的样品呈现较差的稳定性,使用醋酸盐和组氨酸缓冲液处理的样品均呈现较好的稳定性且无明显差异,使用磷酸盐缓冲液处理的样品的稳定性优于使用柠檬酸缓冲液处理的样品的稳定性、略差于使用醋酸盐和组氨酸缓冲液处理的样品的稳定性;在40℃加速实验蛋白定量检测实验中,使用四种缓冲液处理的样品蛋白含量均未出现明显变化。
25℃加速实验反相高效液相色谱和离子交换纯度检测数据均显示,使用柠檬酸缓冲液处理的样品呈现较差的稳定性,使用其它三种缓冲液处理的样品均呈现较好的稳定性且无明显差异;在25℃加速实验蛋白定量检测实验中,使用四种缓冲液处理的样品蛋白含量均未出现明显变化。
从上可知,醋酸盐缓冲液、组氨酸缓冲液以及磷酸盐缓冲液作为缓冲体系时,样品稳定性均较好,均可作为本申请制剂中的缓冲体系。因磷酸盐缓冲液无刺激性,且在多种已上市产品中使用,尤其是当本申请的制剂优选制成吸入剂时,安全性要求更高,故本申请制剂优选使用磷酸盐缓冲液。
实施例11缓冲液浓度筛选
常规称量一水合磷酸二氢钠并用超纯水溶解,NaOH调pH值,分别配制10mmol/L、20mmol/L、40mmol/L、80mmol/L,pH4.5的磷酸盐缓冲液。分别用已配制的不同物质的量浓度的磷酸盐缓冲液进行IL-29突变体蛋白IL-29DE+CS样品的透析处理,透析置换目标缓冲液后将样品使用缓冲液稀配至浓度为300μg/mL,分装0.5mL/支。样品信息如下表26,使用与实施例2中2.2.1相同的方法进行反相高效液相纯度测定,使用与实施例2中2.2.2相同的方法进行离子交换纯度测定,同时使用反相高效液相色谱法进行蛋白定量。
表26
缓冲液浓度筛选加速稳定性条件及取样设计如下表27所示:
表27
40℃加速实验反相高效液相纯度检测结果如下表28所示:
表28
40℃加速实验离子交换纯度检测结果如下表29所示:
表29
40℃加速实验高效液相纯度检测数据显示,使用20~80mmol/L的磷酸盐缓冲液处理的样品呈现较好的稳定性;40℃加速实验离子交换纯度检测数据显示,使用10~40mmol/L的磷酸盐缓冲液处理的样品呈现较好的稳定性;在40℃加速实验蛋白定量检测实验中,使用四种浓度的缓冲液处理的样品蛋白含量均未出现明显变化,说明磷酸盐缓冲液的物质的量浓度在10~80mmol/L范围内时,磷酸盐缓冲液的浓度对样品稳定性的影响差异不大。综上可知,磷酸盐物质的量浓度为20~40mmol/L时,本申请的IL-29突变体IL-29DE+CS的稳定性最好,更优选为20mmol/L。
实施例12 IL-29突变体蛋白制剂
按照下表30中的配方制备各组制剂。首先将辅料磷酸二氢钠一水合物、氯化钠和依地酸二钠二水合物加注射用水溶解后,使用稀盐酸调节溶液pH值至4.5,然后用已配制好的溶液分别将各IL-29突变体蛋白IL-29DE+CS样品进行透析处理,透析置换后将样品稀配至配方浓度待用。使用与实施例2中2.2.1相同的方法进行反相高效液相纯度测定,使用与实施例2中2.2.2相同的方法进行离子交换纯度测定,同时使用反相高效液相色谱法进行蛋白定量。
表30
加速稳定性条件及取样设计如下表31所示:
表31
40℃加速实验反相高效液相纯度检测结果如下表32所示:
表32
40℃加速实验离子交换纯度检测结果如下表33所示:
表33
25℃加速实验反相高效液相纯度检测结果如下表34所示:
表34
25℃光照加速实验离子交换纯度检测结果如下表35所示:
表35
40℃加速实验反相高效液相纯度检测数据显示,相同的IL-29突变体蛋白质量体积浓度下,低依地酸二钠质量体积浓度为0.01~0.05mg/mL的蛋白制剂样品呈现较好的稳定性,尤其是当依地酸二钠质量体积浓度为较低浓度0.01mg/mL时,蛋白制剂的稳定性已经达到比较好的水平。25℃光照加速实验反相高效液相检测数据显示,低依地酸二钠质量体积浓度为0.01mg/mL~0.05mg/mL、且高IL-29突变体蛋白质量体积浓度为0.5~1mg/mL的蛋白制剂的稳定性较好。
由于离子交换色谱方法主要控制杂质为氧化杂质,可以更好的反应依地酸二钠对氧化杂质的抑制效果。40℃加速实验离子交换纯度检测数据显示,低依地酸二钠质量体积浓度为0.01~0.05mg/mL的蛋白制剂样品呈现较好的稳定性,尤其是依地酸二钠质量体积浓度为较低浓度0.01mg/mL,且IL-29突变体蛋白质量体积浓度为0.5~1mg/mL的蛋白制剂的稳定性更好,杂质增长量21天加速控制在了3.962%以下的水平。25℃光照加速实验离子交换纯度检测数据显示,加入依地酸二钠的各组样品氧化杂质增加量均控制在1.0~2.5%以内,而未加依地酸二钠组的杂质增加量在4.5%左右,显示出依地酸二钠对氧化杂质有显著的抑制效果,加入依地酸二钠的蛋白制剂样品的稳定性均明显优于未加入依地酸二钠的蛋白制剂样品。同时,加入依地酸二钠的蛋白制剂样品中依地酸二钠浓度越低杂质增长速度越慢;25℃光照加速实验离子交换纯度检测数据显示,低依地酸二钠质量体积浓度(0.01~0.05mg/mL)、高IL-29突变体蛋白质量体积浓度(0.5~1mg/mL)的蛋白制剂样品呈现较好的稳定性。尤其是依地酸二钠质量体积浓度为0.01mg/mL,且IL-29突变体蛋白质量体积浓度为0.5~1mg/mL的蛋白制剂的稳定性更好,14天和光照加速实验杂质增长量控制在了1.101%以下的水平。
在40℃和25℃加速实验蛋白定量检测实验中,各组蛋白制剂样品的蛋白含量均未出现明显变化。
综合上述结果,优选杂质增长速度较慢、稳定性更高的低依地酸二钠质量体积浓度(0.01~0.05mg/mL)、高IL-29突变体蛋白质量体积浓度(0.5~1mg/mL)的蛋白制剂,尤其是依地酸二钠质量体积浓度为较低浓度0.01mg/mL,IL-29突变体蛋白质量体积浓度为(0.5~1mg/mL)的蛋白制剂。该优选的制剂中,辅料依地酸二钠的加入量可以保持在较低水平,进一步提高了制剂使用的安全性指标。
以上所述,仅是本申请的较佳实施例而已,并非对本申请作其它形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本申请技术方案内容,依据本申请的技术实质对以上实施例所作的任何简单修改、等同变化与改型,仍属于本申请技术方案的保护范围。
序列表:
SEQ ID NO:1
KPTTTGKGC HIGRFKSLSP QELASFKKAR DALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAE LALTLKVLEA AAGPALEDVLDQPLHTLHHI LSQLQACIQPQPTAGPRPRGRLHHWLHRLQEAPKKESAGC LEASVTFNLF RLLTRDLKYV ADGNLCLRTS THPEST
SEQ ID NO:2
GPVPTSKPTTTGKGCHIGRFKSLSPQELASFKKARDALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAELALTLKVLEAAAGPALEDVLDQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQEAPKKESAGCLEASVTFNLFRLLTRDLKYVADGNLCLRTSTHPEST
SEQ ID NO:3
MAAAWTVVLVTLVLGLAVAGPVPTSKPTTTGKGCHIGRFKSLSPQELASFKKARDALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAELALTLKVLEAAAGPALEDVLDQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQEAPKKESAGCLEASVTFNLFRLLTRDLKYVADGNLCLRTSTHPEST
SEQ ID NO:4
MKPTTTGKGC HIGRFKSLSP QELASFKKAR DALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAE LALTLKVLEAAAGPALEDVL DQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQ EAPKKESAGC LEASVTFNLFRLLTRDLKYV AEGNLCLRTSTHPEST
SEQ ID NO:5
MKPTTTGKGC HIGRFKSLSP QELASFKKAR DALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAE LALTLKVLEAAAGPALEDVL DQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQ EAPKKESAGC LEASVTFNLFRLLTRDLKYV AEGNLSLRTSTHPEST
SEQ ID NO:6
MKPTTTGKGC HIGRFKSLSP QELASFKKAR DALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAE LALTLKVLEAAAGPALEDVL DQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQ EAPKKESAGC LEASVTFNLFRLLTRDLKYV ASGNLCLRTSTHPEST
SEQ ID NO:7
MKPTTTGKGC HIGRFKSLSP QELASFKKAR DALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAE LALTLKVLEAAAGPALEDVL DQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQ EAPKKESAGC LEASVTFNLFRLLTRDLKYV ASGNLSLRTSTHPEST
SEQ ID NO:8
MKPTTTGKGC HIGRFKSLSP QELASFKKAR DALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAE LALTLKVLEAAAGPALEDVL DQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQ EAPKKESAGC LEASVTFNLFRLLTRDLKYV ADANLCLRTSTHPEST
SEQ ID NO:9
MKPTTTGKGC HIGRFKSLSP QELASFKKAR DALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAE LALTLKVLEAAAGPALEDVL DQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQ EAPKKESAGC LEASVTFNLFRLLTRDLKYV ADANLSLRTSTHPEST
SEQ ID NO:10,
MKPTTTGKGCHIGRFKSLSP QELASFKKAR DALEESLKLKNWSCSSPVFPGNWDLRLLQVRERPVALEAE LALTLKVLEAAAGPALEDVL DQPLHTLHHILSQLQACIQPQPTAGPRPRGRLHHWLHRLQ EAPKKESAGC LEASVTFNLFRLLTRDLKYV ADGNLSLRTSTHPEST
SEQ ID NO:11
SEQ ID NO:12
SEQ ID NO:13
SEQ ID NO:14
SEQ ID NO:15
SEQ ID NO:16
SEQ ID NO:17
SEQ ID NO:18
序列表
<110> 杭州先为达生物科技有限公司
<120> 一种白细胞介素29突变体蛋白制剂
<130> PD01241
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 175
<212> PRT
<213> Artificial Sequence
<400> 1
Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys Ser
1 5 10 15
Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala Leu
20 25 30
Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val Phe
35 40 45
Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro Val
50 55 60
Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala Ala
65 70 75 80
Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr Leu
85 90 95
His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro Thr
100 105 110
Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg Leu
115 120 125
Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser Val
130 135 140
Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val Ala
145 150 155 160
Asp Gly Asn Leu Cys Leu Arg Thr Ser Thr His Pro Glu Ser Thr
165 170 175
<210> 2
<211> 181
<212> PRT
<213> Artificial Sequence
<400> 2
Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys Gly Cys His
1 5 10 15
Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys
20 25 30
Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser
35 40 45
Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln
50 55 60
Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu
65 70 75 80
Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp
85 90 95
Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu Gln Ala Cys
100 105 110
Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His
115 120 125
His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly
130 135 140
Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg
145 150 155 160
Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg Thr Ser Thr
165 170 175
His Pro Glu Ser Thr
180
<210> 3
<211> 200
<212> PRT
<213> Artificial Sequence
<400> 3
Met Ala Ala Ala Trp Thr Val Val Leu Val Thr Leu Val Leu Gly Leu
1 5 10 15
Ala Val Ala Gly Pro Val Pro Thr Ser Lys Pro Thr Thr Thr Gly Lys
20 25 30
Gly Cys His Ile Gly Arg Phe Lys Ser Leu Ser Pro Gln Glu Leu Ala
35 40 45
Ser Phe Lys Lys Ala Arg Asp Ala Leu Glu Glu Ser Leu Lys Leu Lys
50 55 60
Asn Trp Ser Cys Ser Ser Pro Val Phe Pro Gly Asn Trp Asp Leu Arg
65 70 75 80
Leu Leu Gln Val Arg Glu Arg Pro Val Ala Leu Glu Ala Glu Leu Ala
85 90 95
Leu Thr Leu Lys Val Leu Glu Ala Ala Ala Gly Pro Ala Leu Glu Asp
100 105 110
Val Leu Asp Gln Pro Leu His Thr Leu His His Ile Leu Ser Gln Leu
115 120 125
Gln Ala Cys Ile Gln Pro Gln Pro Thr Ala Gly Pro Arg Pro Arg Gly
130 135 140
Arg Leu His His Trp Leu His Arg Leu Gln Glu Ala Pro Lys Lys Glu
145 150 155 160
Ser Ala Gly Cys Leu Glu Ala Ser Val Thr Phe Asn Leu Phe Arg Leu
165 170 175
Leu Thr Arg Asp Leu Lys Tyr Val Ala Asp Gly Asn Leu Cys Leu Arg
180 185 190
Thr Ser Thr His Pro Glu Ser Thr
195 200
<210> 4
<211> 176
<212> PRT
<213> Artificial Sequence
<400> 4
Met Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys
1 5 10 15
Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala
20 25 30
Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val
35 40 45
Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro
50 55 60
Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala
65 70 75 80
Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr
85 90 95
Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro
100 105 110
Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg
115 120 125
Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser
130 135 140
Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val
145 150 155 160
Ala Glu Gly Asn Leu Cys Leu Arg Thr Ser Thr His Pro Glu Ser Thr
165 170 175
<210> 5
<211> 176
<212> PRT
<213> Artificial Sequence
<400> 5
Met Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys
1 5 10 15
Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala
20 25 30
Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val
35 40 45
Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro
50 55 60
Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala
65 70 75 80
Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr
85 90 95
Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro
100 105 110
Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg
115 120 125
Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser
130 135 140
Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val
145 150 155 160
Ala Glu Gly Asn Leu Ser Leu Arg Thr Ser Thr His Pro Glu Ser Thr
165 170 175
<210> 6
<211> 176
<212> PRT
<213> Artificial Sequence
<400> 6
Met Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys
1 5 10 15
Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala
20 25 30
Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val
35 40 45
Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro
50 55 60
Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala
65 70 75 80
Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr
85 90 95
Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro
100 105 110
Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg
115 120 125
Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser
130 135 140
Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val
145 150 155 160
Ala Ser Gly Asn Leu Cys Leu Arg Thr Ser Thr His Pro Glu Ser Thr
165 170 175
<210> 7
<211> 176
<212> PRT
<213> Artificial Sequence
<400> 7
Met Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys
1 5 10 15
Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala
20 25 30
Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val
35 40 45
Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro
50 55 60
Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala
65 70 75 80
Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr
85 90 95
Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro
100 105 110
Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg
115 120 125
Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser
130 135 140
Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val
145 150 155 160
Ala Ser Gly Asn Leu Ser Leu Arg Thr Ser Thr His Pro Glu Ser Thr
165 170 175
<210> 8
<211> 176
<212> PRT
<213> Artificial Sequence
<400> 8
Met Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys
1 5 10 15
Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala
20 25 30
Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val
35 40 45
Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro
50 55 60
Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala
65 70 75 80
Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr
85 90 95
Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro
100 105 110
Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg
115 120 125
Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser
130 135 140
Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val
145 150 155 160
Ala Asp Ala Asn Leu Cys Leu Arg Thr Ser Thr His Pro Glu Ser Thr
165 170 175
<210> 9
<211> 176
<212> PRT
<213> Artificial Sequence
<400> 9
Met Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys
1 5 10 15
Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala
20 25 30
Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val
35 40 45
Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro
50 55 60
Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala
65 70 75 80
Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr
85 90 95
Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro
100 105 110
Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg
115 120 125
Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser
130 135 140
Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val
145 150 155 160
Ala Asp Ala Asn Leu Ser Leu Arg Thr Ser Thr His Pro Glu Ser Thr
165 170 175
<210> 10
<211> 176
<212> PRT
<213> Artificial Sequence
<400> 10
Met Lys Pro Thr Thr Thr Gly Lys Gly Cys His Ile Gly Arg Phe Lys
1 5 10 15
Ser Leu Ser Pro Gln Glu Leu Ala Ser Phe Lys Lys Ala Arg Asp Ala
20 25 30
Leu Glu Glu Ser Leu Lys Leu Lys Asn Trp Ser Cys Ser Ser Pro Val
35 40 45
Phe Pro Gly Asn Trp Asp Leu Arg Leu Leu Gln Val Arg Glu Arg Pro
50 55 60
Val Ala Leu Glu Ala Glu Leu Ala Leu Thr Leu Lys Val Leu Glu Ala
65 70 75 80
Ala Ala Gly Pro Ala Leu Glu Asp Val Leu Asp Gln Pro Leu His Thr
85 90 95
Leu His His Ile Leu Ser Gln Leu Gln Ala Cys Ile Gln Pro Gln Pro
100 105 110
Thr Ala Gly Pro Arg Pro Arg Gly Arg Leu His His Trp Leu His Arg
115 120 125
Leu Gln Glu Ala Pro Lys Lys Glu Ser Ala Gly Cys Leu Glu Ala Ser
130 135 140
Val Thr Phe Asn Leu Phe Arg Leu Leu Thr Arg Asp Leu Lys Tyr Val
145 150 155 160
Ala Asp Gly Asn Leu Ser Leu Arg Thr Ser Thr His Pro Glu Ser Thr
165 170 175
<210> 11
<211> 528
<212> DNA
<213> Artificial Sequence
<400> 11
atgaaaccga ccacgaccgg caaaggctgc catattggtc gctttaagtc gctgtcgccg 60
caggaactgg cgagcttcaa gaaagcccgt gatgccctgg aggaatcgct gaaactgaag 120
aactggagct gtagctcgcc ggtgttcccg ggcaactggg atctgcgtct gctgcaggtt 180
cgcgaacgtc cggttgcgct ggaagcggaa ctggcgctga ccctgaaagt gctggaagcg 240
gcagcgggtc cggcgctgga agatgttctg gatcagccgc tgcacaccct gcatcatatt 300
ctgtcgcagc tgcaggcgtg cattcaaccg cagccgaccg cgggcccgcg tccgcgcggc 360
cgtctgcatc actggctgca ccgtctgcag gaagccccga agaaagagtc ggcgggctgt 420
ctggaagcgt cggtgacctt caatctgttc cgtctgctga cccgtgatct gaaatacgtt 480
gcggaaggca atctgtctct gcgtacctcg acccacccgg aatcgacc 544
<210> 12
<211> 537
<212> DNA
<213> Artificial Sequence
<400> 12
atgaaaccga ccacgaccgg caaaggctgc catattggtc gctttaagtc gctgtcgccg 60
caggaactgg cgagcttcaa gaaagcccgt gatgccctgg aggaatcgct gaaactgaag 120
aactggagct gtagctcgcc ggtgttcccg ggcaactggg atctgcgtct gctgcaggtt 180
cgcgaacgtc cggttgcgct ggaagcggaa ctggcgctga ccctgaaagt gctggaagcg 240
gcagcgggtc cggcgctgga agatgttctg gatcagccgc tgcacaccct gcatcatatt 300
ctgtcgcagc tgcaggcgtg cattcaaccg cagccgaccg cgggcccgcg tccgcgcggc 360
cgtctgcatc actggctgca ccgtctgcag gaagccccga agaaagagtc ggcgggctgt 420
ctggaagcgt cggtgacctt caatctgttc cgtctgctga cccgtgatct gaaatacgtt 480
gcgagcggca atctgtctct gcgtacctcg acccacccgg aatcgaccta ataataa 553
<210> 13
<211> 537
<212> DNA
<213> Artificial Sequence
<400> 13
atgaaaccga ccacgaccgg caaaggctgc catattggtc gctttaagtc gctgtcgccg 60
caggaactgg cgagcttcaa gaaagcccgt gatgccctgg aggaatcgct gaaactgaag 120
aactggagct gtagctcgcc ggtgttcccg ggcaactggg atctgcgtct gctgcaggtt 180
cgcgaacgtc cggttgcgct ggaagcggaa ctggcgctga ccctgaaagt gctggaagcg 240
gcagcgggtc cggcgctgga agatgttctg gatcagccgc tgcacaccct gcatcatatt 300
ctgtcgcagc tgcaggcgtg cattcaaccg cagccgaccg cgggcccgcg tccgcgcggc 360
cgtctgcatc actggctgca ccgtctgcag gaagccccga agaaagagtc ggcgggctgt 420
ctggaagcgt cggtgacctt caatctgttc cgtctgctga cccgtgatct gaaatacgtt 480
gcggacgcga atctgtctct gcgtacctcg acccacccgg aatcgaccta ataataa 553
<210> 14
<211> 528
<212> DNA
<213> Artificial Sequence
<400> 14
atgaaaccga ccacgaccgg caaaggctgc catattggtc gctttaagtc gctgtcgccg 60
caggaactgg cgagcttcaa gaaagcccgt gatgccctgg aggaatcgct gaaactgaag 120
aactggagct gtagctcgcc ggtgttcccg ggcaactggg atctgcgtct gctgcaggtt 180
cgcgaacgtc cggttgcgct ggaagcggaa ctggcgctga ccctgaaagt gctggaagcg 240
gcagcgggtc cggcgctgga agatgttctg gatcagccgc tgcacaccct gcatcatatt 300
ctgtcgcagc tgcaggcgtg cattcaaccg cagccgaccg cgggcccgcg tccgcgcggc 360
cgtctgcatc actggctgca ccgtctgcag gaagccccga agaaagagtc ggcgggctgt 420
ctggaagcgt cggtgacctt caatctgttc cgtctgctga cccgtgatct gaaatacgtt 480
gcggaaggca atctgtgtct gcgtacctcg acccacccgg aatcgacc 544
<210> 15
<211> 537
<212> DNA
<213> Artificial Sequence
<400> 15
atgaaaccga ccacgaccgg caaaggctgc catattggtc gctttaagtc gctgtcgccg 60
caggaactgg cgagcttcaa gaaagcccgt gatgccctgg aggaatcgct gaaactgaag 120
aactggagct gtagctcgcc ggtgttcccg ggcaactggg atctgcgtct gctgcaggtt 180
cgcgaacgtc cggttgcgct ggaagcggaa ctggcgctga ccctgaaagt gctggaagcg 240
gcagcgggtc cggcgctgga agatgttctg gatcagccgc tgcacaccct gcatcatatt 300
ctgtcgcagc tgcaggcgtg cattcaaccg cagccgaccg cgggcccgcg tccgcgcggc 360
cgtctgcatc actggctgca ccgtctgcag gaagccccga agaaagagtc ggcgggctgt 420
ctggaagcgt cggtgacctt caatctgttc cgtctgctga cccgtgatct gaaatacgtt 480
gcgagcggca atctgtgtct gcgtacctcg acccacccgg aatcgaccta ataataa 553
<210> 16
<211> 537
<212> DNA
<213> Artificial Sequence
<400> 16
atgaaaccga ccacgaccgg caaaggctgc catattggtc gctttaagtc gctgtcgccg 60
caggaactgg cgagcttcaa gaaagcccgt gatgccctgg aggaatcgct gaaactgaag 120
aactggagct gtagctcgcc ggtgttcccg ggcaactggg atctgcgtct gctgcaggtt 180
cgcgaacgtc cggttgcgct ggaagcggaa ctggcgctga ccctgaaagt gctggaagcg 240
gcagcgggtc cggcgctgga agatgttctg gatcagccgc tgcacaccct gcatcatatt 300
ctgtcgcagc tgcaggcgtg cattcaaccg cagccgaccg cgggcccgcg tccgcgcggc 360
cgtctgcatc actggctgca ccgtctgcag gaagccccga agaaagagtc ggcgggctgt 420
ctggaagcgt cggtgacctt caatctgttc cgtctgctga cccgtgatct gaaatacgtt 480
gcggacgcga atctgtgtct gcgtacctcg acccacccgg aatcgaccta ataataa 553
<210> 17
<211> 537
<212> DNA
<213> Artificial Sequence
<400> 17
atgaaaccga ccacgaccgg caaaggctgc catattggtc gctttaagtc gctgtcgccg 60
caggaactgg cgagcttcaa gaaagcccgt gatgccctgg aggaatcgct gaaactgaag 120
aactggagct gtagctcgcc ggtgttcccg ggcaactggg atctgcgtct gctgcaggtt 180
cgcgaacgtc cggttgcgct ggaagcggaa ctggcgctga ccctgaaagt gctggaagcg 240
gcagcgggtc cggcgctgga agatgttctg gatcagccgc tgcacaccct gcatcatatt 300
ctgtcgcagc tgcaggcgtg cattcaaccg cagccgaccg cgggcccgcg tccgcgcggc 360
cgtctgcatc actggctgca ccgtctgcag gaagccccga agaaagagtc ggcgggctgt 420
ctggaagcgt cggtgacctt caatctgttc cgtctgctga cccgtgatct gaaatacgtt 480
gcggacggca atctgtctct gcgtacctcg acccacccgg aatcgaccta ataataa 553
<210> 18
<211> 534
<212> DNA
<213> Artificial Sequence
<400> 18
aaaccgacca cgaccggcaa aggctgccat attggtcgct ttaagtcgct gtcgccgcag 60
gaactggcga gcttcaagaa agcccgtgat gccctggagg aatcgctgaa actgaagaac 120
tggagctgta gctcgccggt gttcccgggc aactgggatc tgcgtctgct gcaggttcgc 180
gaacgtccgg ttgcgctgga agcggaactg gcgctgaccc tgaaagtgct ggaagcggca 240
gcgggtccgg cgctggaaga tgttctggat cagccgctgc acaccctgca tcatattctg 300
tcgcagctgc aggcgtgcat tcaaccgcag ccgaccgcgg gcccgcgtcc gcgcggccgt 360
ctgcatcact ggctgcaccg tctgcaggaa gccccgaaga aagagtcggc gggctgtctg 420
gaagcgtcgg tgaccttcaa tctgttccgt ctgctgaccc gtgatctgaa atacgttgcg 480
gacggcaatc tgtgtctgcg tacctcgacc cacccggaat cgacctaata ataa 550
Claims (10)
1.一种白细胞介素29突变体蛋白制剂,其特征在于,其包含:白细胞介素29突变体蛋白、缓冲体系、金属离子螯合剂和渗透压调节剂;
其中,所述白细胞介素29突变体蛋白的质量体积浓度为0.1~1mg/mL;
所述缓冲体系为磷酸盐缓冲液,所述磷酸盐缓冲液的物质的量浓度为20~80mmol/L;
所述金属离子螯合剂为依地酸二钠,所述依地酸二钠的质量体积浓度为0.01~0.1mg/mL;
所述渗透压调节剂为氯化钠;
所述制剂的pH为4.0~5.0;
所述白细胞介素29突变体蛋白包含SEQ ID NO:1所示氨基酸序列上第161或第162位氨基酸的取代突变,其中所述第161位的天冬氨酸(D)或者第162位的甘氨酸(G)被其它天然氨基酸取代。
2.根据权利要求1所述的制剂,其特征在于,所述第161位的天冬氨酸(D)被谷氨酸、苏氨酸或丝氨酸取代,或者第162位的甘氨酸(G)被脂肪族氨基酸取代。
3.根据权利要求1所述的制剂,其特征在于,所述白细胞介素29突变体蛋白包含如下的氨基酸序列:SEQ ID NO:4,SEQ ID NO:6或SEQ ID NO:8。
4.根据权利要求1所述的制剂,其特征在于,所述白细胞介素29突变体蛋白还包含SEQID NO:1所示氨基酸序列上第165位从半胱氨酸(C)到丝氨酸(S)的取代突变。
5.根据权利要求1所述的制剂,其特征在于,所述白细胞介素29突变体蛋白包含如下的氨基酸序列:SEQ ID NO:5,SEQ ID NO:7或SEQ ID NO:9。
6.根据权利要求1~5中任一项所述的制剂,其特征在于,所述制剂的pH为4.0~4.5。
7.根据权利要求1~5中任一项所述的制剂,其特征在于,所述磷酸盐缓冲液的物质的量浓度为20~40mmol/L。
8.根据权利要求1~5中任一项所述的制剂,其特征在于,所述依地酸二钠的质量体积浓度为0.01~0.05mg/mL。
9.根据权利要求1~5中任一项所述的制剂,其特征在于,所述氯化钠的质量体积浓度为6~10mg/mL。
10.根据权利要求1~5中任一项所述的制剂,其特征在于,所述白细胞介素29突变体蛋白的质量体积浓度为0.5~1mg/mL,所述依地酸二钠的质量体积浓度为0.01mg/mL。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110206470.3A CN112870336B (zh) | 2021-02-24 | 2021-02-24 | 一种白细胞介素29突变体蛋白制剂 |
PCT/CN2021/080451 WO2022178922A1 (zh) | 2021-02-24 | 2021-03-12 | 一种白细胞介素29突变体蛋白制剂 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110206470.3A CN112870336B (zh) | 2021-02-24 | 2021-02-24 | 一种白细胞介素29突变体蛋白制剂 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN112870336A CN112870336A (zh) | 2021-06-01 |
CN112870336B true CN112870336B (zh) | 2022-06-14 |
Family
ID=76054741
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110206470.3A Active CN112870336B (zh) | 2021-02-24 | 2021-02-24 | 一种白细胞介素29突变体蛋白制剂 |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN112870336B (zh) |
WO (1) | WO2022178922A1 (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113956347B (zh) * | 2021-12-13 | 2023-05-26 | 青岛瑞斯凯尔生物科技有限公司 | Il-8突变体及其应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105085658A (zh) * | 2014-05-14 | 2015-11-25 | 北京凯因科技股份有限公司 | 一种白细胞介素29突变体及聚乙二醇衍生物 |
CN105816857A (zh) * | 2016-04-22 | 2016-08-03 | 北京三元基因药业股份有限公司 | 干扰素的水溶液制剂 |
CN107698682A (zh) * | 2010-05-03 | 2018-02-16 | 百时美施贵宝公司 | 血清白蛋白结合分子 |
CN109293782A (zh) * | 2014-01-08 | 2019-02-01 | 德益阳光生物技术(北京)有限责任公司 | 融合多肽及使用方法 |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2574564C (en) * | 2004-07-29 | 2013-04-16 | Zymogenetics, Inc. | Use of il-28 and il-29 to treat cancer and autoimmune disorders |
US9272029B2 (en) * | 2009-03-26 | 2016-03-01 | Ibc Pharmaceuticals, Inc. | Interferon lambada-antibody complexes |
CN106913869B (zh) * | 2017-03-17 | 2020-07-28 | 信达生物制药(苏州)有限公司 | 一种抗ctla-4单克隆抗体制剂及其应用 |
KR20210024082A (ko) * | 2018-06-25 | 2021-03-04 | 제이씨알 파마 가부시키가이샤 | 단백질 함유 수성 액제 |
-
2021
- 2021-02-24 CN CN202110206470.3A patent/CN112870336B/zh active Active
- 2021-03-12 WO PCT/CN2021/080451 patent/WO2022178922A1/zh active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107698682A (zh) * | 2010-05-03 | 2018-02-16 | 百时美施贵宝公司 | 血清白蛋白结合分子 |
CN109293782A (zh) * | 2014-01-08 | 2019-02-01 | 德益阳光生物技术(北京)有限责任公司 | 融合多肽及使用方法 |
CN105085658A (zh) * | 2014-05-14 | 2015-11-25 | 北京凯因科技股份有限公司 | 一种白细胞介素29突变体及聚乙二醇衍生物 |
CN105816857A (zh) * | 2016-04-22 | 2016-08-03 | 北京三元基因药业股份有限公司 | 干扰素的水溶液制剂 |
Non-Patent Citations (2)
Title |
---|
TIINA LIPIAINEN等.Formulation and Stability of Cytokine Therapeutics.《JOURNAL OF PHARMACEUTICAL SCIENCES》.2014, * |
WILLIAM GARZON-RODRIGUEZ等.Optimizing Storage Stability of Lyophilized Recombinant Human Interleukin-11 with Disaccharide/Hydroxyethyl Starch Mixtures.《JOURNAL OF PHARMACEUTICAL SCIENCES》.2004, * |
Also Published As
Publication number | Publication date |
---|---|
WO2022178922A1 (zh) | 2022-09-01 |
CN112870336A (zh) | 2021-06-01 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20110189128A1 (en) | Recombinant super-compound interferon | |
RU2144374C1 (ru) | Способ получения композита окисленного глутатиона с cis-диаминодихлорплатиной и фармацевтических композиций на его основе, регулирующих метаболизм, пролиферацию, дифференцировку и механизмы апоптоза нормальных и трансформированных клеток | |
CN106632682A (zh) | 融合蛋白ifn-elp及其应用 | |
WO2015161820A1 (zh) | 两亲性合成抗菌肽、其药物组合物及其用途 | |
CN113368234B (zh) | 一种稳定的抗csf-1r单克隆抗体的液体制剂及应用 | |
US20190352345A1 (en) | Mutants of recombinant immunoregulatory protein of Ganoderma lucidum and applications thereof | |
CN112870336B (zh) | 一种白细胞介素29突变体蛋白制剂 | |
CN102655876B (zh) | α-1蛋白酶抑制剂组合物、方法和试剂盒 | |
EP1228097A2 (en) | Antimicrobial activity of the first cationic cluster of human lactoferrin | |
TW202241492A (zh) | Glp-1/glp-2雙重促效劑之醫藥組合物 | |
EP3173420A1 (en) | Polypeptide and polypeptide complex for suppressing tumor metastasis and treating leukemia as well as preparation method therefor and application thereof | |
US6372890B1 (en) | Water-soluble polypeptides | |
CN113769081A (zh) | 一种稳定的高浓度抗人il-5单克隆抗体液体制剂 | |
CN112694526B (zh) | 一种白细胞介素29突变体蛋白 | |
US10988429B2 (en) | Quinochalcone compound and uses thereof for treating cancer or inflammation | |
CN101781369A (zh) | 一种重组人精氨酸酶融合蛋白及其应用 | |
KR102291913B1 (ko) | Hgf의 동결건조 제제 | |
JP2023543496A (ja) | 血管透過性の問題に対処するオートファジー阻害ペプチド及びその有機酸塩 | |
AU2021280623B9 (en) | Interleukin 29 mutant protein | |
CN115385998A (zh) | 稳定的iii型干扰素蛋白及其融合蛋白 | |
CN111825773B (zh) | 重组人TSG6-IFNα融合蛋白及其制备方法以及其作为抗病毒药物的应用 | |
CN112979764B (zh) | 特异结合人cd47分子的多肽及其用途 | |
JPH06247870A (ja) | インターロイキン−6を含有する医薬製剤 | |
WO2024050300A2 (en) | Synthetic surfactants for inhibiting coronavirus infection | |
US5693754A (en) | Inhibitory binding protein for insulin-like growth factors |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
CP03 | Change of name, title or address |
Address after: 310018 Room 1802, Building 2, Hangzhou East International Business Center, Qiantang District, Hangzhou City, Zhejiang Province Patentee after: Hangzhou Xianweida Biotechnology Co.,Ltd. Address before: Room 815-818, building 17, No.57, kejiyuan Road, Baiyang street, Hangzhou Economic and Technological Development Zone, Hangzhou City, Zhejiang Province 310018 Patentee before: HANGZHOU SCIWIND BIOSCIENCES Co.,Ltd. |
|
CP03 | Change of name, title or address |