CN103694339B - A kind of refolding method of insulin glargine precursor - Google Patents
A kind of refolding method of insulin glargine precursor Download PDFInfo
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- CN103694339B CN103694339B CN201310754124.4A CN201310754124A CN103694339B CN 103694339 B CN103694339 B CN 103694339B CN 201310754124 A CN201310754124 A CN 201310754124A CN 103694339 B CN103694339 B CN 103694339B
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- 108010057186 Insulin Glargine Proteins 0.000 title claims abstract description 105
- COCFEDIXXNGUNL-RFKWWTKHSA-N Insulin glargine Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3NC=NC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(=O)NCC(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1=CN=CN1 COCFEDIXXNGUNL-RFKWWTKHSA-N 0.000 title claims abstract description 105
- 229960002869 insulin glargine Drugs 0.000 title claims abstract description 105
- 239000002243 precursor Substances 0.000 title claims abstract description 95
- 238000000034 method Methods 0.000 title claims abstract description 39
- 239000000243 solution Substances 0.000 claims abstract description 72
- 238000004153 renaturation Methods 0.000 claims abstract description 60
- 238000006243 chemical reaction Methods 0.000 claims abstract description 40
- 239000003638 chemical reducing agent Substances 0.000 claims abstract description 24
- 230000033228 biological regulation Effects 0.000 claims abstract description 17
- 239000013024 dilution buffer Substances 0.000 claims abstract description 16
- 230000012846 protein folding Effects 0.000 claims abstract description 16
- 239000000654 additive Substances 0.000 claims abstract description 14
- 239000003398 denaturant Substances 0.000 claims abstract description 13
- 238000004925 denaturation Methods 0.000 claims abstract description 13
- 230000036425 denaturation Effects 0.000 claims abstract description 13
- 230000000996 additive effect Effects 0.000 claims abstract description 12
- 230000035484 reaction time Effects 0.000 claims abstract description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 39
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 claims description 38
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 28
- 235000011187 glycerol Nutrition 0.000 claims description 19
- 229910021645 metal ion Inorganic materials 0.000 claims description 18
- 239000002202 Polyethylene glycol Substances 0.000 claims description 16
- 229920001223 polyethylene glycol Polymers 0.000 claims description 16
- 239000004471 Glycine Substances 0.000 claims description 14
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 claims description 14
- 239000004202 carbamide Substances 0.000 claims description 12
- XSQUKJJJFZCRTK-UHFFFAOYSA-N urea group Chemical group NC(=O)N XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 12
- 239000007853 buffer solution Substances 0.000 claims description 11
- 235000009508 confectionery Nutrition 0.000 claims description 10
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 8
- 229920001030 Polyethylene Glycol 4000 Polymers 0.000 claims description 7
- QIJRTFXNRTXDIP-UHFFFAOYSA-N (1-carboxy-2-sulfanylethyl)azanium;chloride;hydrate Chemical compound O.Cl.SCC(N)C(O)=O QIJRTFXNRTXDIP-UHFFFAOYSA-N 0.000 claims description 6
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 6
- 108090001061 Insulin Proteins 0.000 claims description 6
- 102000004877 Insulin Human genes 0.000 claims description 6
- 229960001305 cysteine hydrochloride Drugs 0.000 claims description 6
- 229960000789 guanidine hydrochloride Drugs 0.000 claims description 6
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 6
- 229940125396 insulin Drugs 0.000 claims description 6
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 4
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 claims description 3
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 claims description 3
- 239000008118 PEG 6000 Substances 0.000 claims description 3
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 claims description 3
- 235000012538 ammonium bicarbonate Nutrition 0.000 claims description 3
- 239000001099 ammonium carbonate Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000004254 Ammonium phosphate Substances 0.000 claims description 2
- 229910000148 ammonium phosphate Inorganic materials 0.000 claims description 2
- 235000019289 ammonium phosphates Nutrition 0.000 claims description 2
- 239000006193 liquid solution Substances 0.000 claims 1
- 238000000137 annealing Methods 0.000 abstract description 6
- 239000003814 drug Substances 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 16
- 108090000623 proteins and genes Proteins 0.000 description 14
- 235000018102 proteins Nutrition 0.000 description 12
- 102000004169 proteins and genes Human genes 0.000 description 12
- 210000003000 inclusion body Anatomy 0.000 description 10
- 230000008569 process Effects 0.000 description 8
- 241000588724 Escherichia coli Species 0.000 description 7
- 210000000496 pancreas Anatomy 0.000 description 6
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 5
- YAJCHEVQCOHZDC-QMMNLEPNSA-N actrapid Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3N=CNC=3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@H](C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@H](C)CC)[C@H](C)CC)[C@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C(N)=O)C1=CNC=N1 YAJCHEVQCOHZDC-QMMNLEPNSA-N 0.000 description 5
- LEMUFSYUPGXXCM-JNEQYSBXSA-N caninsulin Chemical compound [Zn].C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@H]1CSSC[C@H]2C(=O)N[C@H](C(=O)N[C@@H](CO)C(=O)N[C@H](C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=3C=CC(O)=CC=3)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=3C=CC(O)=CC=3)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC3N=CN=C3)NC(=O)[C@H](CO)NC(=O)CNC1=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)O)C(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O)=O)CSSC[C@@H](C(N2)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](NC(=O)CN)[C@@H](C)CC)[C@@H](C)CC)[C@@H](C)O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)CC=1C=CC=CC=1)C(C)C)C1C=NC=N1 LEMUFSYUPGXXCM-JNEQYSBXSA-N 0.000 description 5
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 239000004475 Arginine Substances 0.000 description 4
- 229910021592 Copper(II) chloride Inorganic materials 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 230000002776 aggregation Effects 0.000 description 4
- 238000004220 aggregation Methods 0.000 description 4
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 238000010606 normalization Methods 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 108020004511 Recombinant DNA Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical compound Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 description 3
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000003647 oxidation Effects 0.000 description 3
- 238000007254 oxidation reaction Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 108010075254 C-Peptide Proteins 0.000 description 2
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 229960002433 cysteine Drugs 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- -1 cysteine hydrochlorides Chemical class 0.000 description 2
- 229960003067 cystine Drugs 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- TZBAVQKIEKDGFH-UHFFFAOYSA-N n-[2-(diethylamino)ethyl]-1-benzothiophene-2-carboxamide;hydrochloride Chemical compound [Cl-].C1=CC=C2SC(C(=O)NCC[NH+](CC)CC)=CC2=C1 TZBAVQKIEKDGFH-UHFFFAOYSA-N 0.000 description 2
- 230000001590 oxidative effect Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-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
- 229910021591 Copper(I) chloride Inorganic materials 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- 208000013016 Hypoglycemia Diseases 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
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108010076181 Proinsulin Proteins 0.000 description 1
- 102000007327 Protamines Human genes 0.000 description 1
- 108010007568 Protamines Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000003196 chaotropic effect Effects 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- OXBLHERUFWYNTN-UHFFFAOYSA-M copper(I) chloride Chemical compound [Cu]Cl OXBLHERUFWYNTN-UHFFFAOYSA-M 0.000 description 1
- 230000009615 deamination Effects 0.000 description 1
- 238000006481 deamination reaction Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000009322 erkang Substances 0.000 description 1
- 239000000686 essence Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000004026 insulin derivative Substances 0.000 description 1
- 230000003914 insulin secretion Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000003182 parenteral nutrition solution Substances 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 229940057838 polyethylene glycol 4000 Drugs 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 229940048914 protamine Drugs 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/62—Insulins
Abstract
The invention discloses a kind of refolding method of insulin glargine precursor, belong to biological medicine protein folding field.The present invention adds reducing agent and reduced by the way that insulin glargine precursor is dissolved in denaturant solution, and regulation pH value is 9.5~11.5, and the temperature for controlling reaction system is 35 DEG C~45 DEG C, reacts 30~60min, the insulin glargine precursor solution after being denatured；Insulin glargine precursor after denaturation is added in dilution buffer, protein folding additive is added, regulation pH value is 9.5~11.5, air is continually fed into solution, 0 DEG C~20 DEG C of the temperature of reaction system is controlled, is reacted 2~40 hours, insulin glargine renaturation solution is obtained.Present invention reduces the renaturation reaction time, the protein content correctly folded is improved, annealing efficiency is improved to 51%~62%, while reducing production cost, be adapted to industrialized production application.
Description
Technical field
The invention belongs to biological medicine protein folding field, and in particular to a kind of refolding method of insulin glargine precursor,
More particularly to a kind of air oxidation dilution by the use of polyethylene glycol, glycerine and metal ion etc. as protein folding additive is multiple
Property method, changes into the precursor with correct space structure, annealing efficiency can surpass by the insulin glargine precursor of false folding
50% is crossed, the generation of disulfide bond mispairing accessory substance is reduced, brought great convenience to subsequent purification work.
Background technology
Insulin glargine（glycine arginine insulin）It is that one kind is produced by recombinant DNA technology and is used to control
Treat I, the protamine zine insulin kind biological product of type II diabetes.It is to add two smart ammonia in actrapid monotard's B chain carboxyl terminals
Acid, while the asparagine of A chain carboxyl terminal A21 positions is also substituted for glycine, A chain carboxyl terminals prevent for glycine
The formation of deamination products, makes insulin glargine have good stability；Again because increased two arginine are positively charged, institute
With the isoelectric point of insulin glargine by 5.4（Insulin isoelectric point）Left and right is increased to 7.0 or so, therefore the insulin analog can
With under conditions of meta-acid（pH4.0）It is configured to the parenteral solution that property is stable and clarifies.Do not need to be suspended before insulin glargine injection,
After hypodermic injection in vivo（pH7.4）The sustainable slow release insulin glargine of microscopic precipitate is formed, plasma concentration is steady, peak valley
Curve is small, and acting duration is long.A blood sugar reducing function is subcutaneously injected daily can maintain 24 hours, and without obvious blood medicine peak value
Occur, can preferable simulation arm's length basis insulin secretion, the probability of reduction generation hypoglycemia.
The insulin glargine precursor produced both at home and abroad at present uses Bacillus coli expression, and eucaryote shape is lacked in its system
Into the environment and the modified elements of correlation of the correct structure of albumen, high level expression frequently results in albumen aggregation in Escherichia coli
And form insoluble, inactive inclusion body.Inclusion body is formed with bioactivity after separation, denaturation dissolving and renaturation
Albumen, needs to be provided with the folding environment beneficial to single chain protein in renaturation process, while appropriate redox environment is provided,
Insulin glargine precursor is set to form correct secondary structure.
The efficiency of renaturation directly affects the height of yield.Renaturation is the albumen in a complicated process, renaturation process
Aggreation is the first cause for causing renaturation yield low.After the concentration of denaturant is relatively low, single-stranded protein molecular is folded rapidly
The intermediate state molecule of substantial amounts of secondary structure is formed, the hydrophobic region interaction of intermediate state molecular surface exposure occurs aggregation and made
With.The aggregation of albumen and it is correct it is collapsible between be competitive reaction, therefore reduce the aggtegation of albumen, improve the correct of albumen
Fold most important to renaturation.
The refolding method of insulin is related in the prior art, such as, in F-J Lu Bailuode C07K14/62, China is specially
The improved method for obtaining the insulin precurosor with correctly bonded cystine linkage is disclosed in sharp CN1132845C, i.e., half
In the presence of cystine or cysteine hydrochloride and chaotropic auxiliary agent, 30%~50% correct renaturation product is acquired.Wang Li is beautiful
A kind of Efficient numerical method renaturation Simultaneous purification recombined human is disclosed in C07K14/62, Chinese patent CN103172727A
The method of proinsulin, attempts this method in insulin glargine precursor carries out renaturation, as a result shows that the rate of recovery is low.At present, exist
Rarely has technique and the parameter report on the high annealing efficiency of insulin glargine in disclosed document.
The content of the invention
It is an object of the invention to by optimizing renaturation technological parameter, there is provided a kind of renaturation side of insulin glargine precursor
Method, i.e., dissolve inclusion body with denaturant, the nonactive disulfide bond added in the disulfide bond and chain of reducing agent opening interchain formation, tool
The insulin glargine precursor for having primary structure is properly fold into active albumen in dilution, is added in renaturation process
The protein folding additive such as polyethylene glycol, glycerine, metal ion.This method can improve the efficiency of renaturation to 51%~62%, drop
The low generation of disulfide bond mispairing accessory substance, brings great convenience to subsequent purification work.
The purpose of the present invention is realized by using following technical proposals：A kind of renaturation side of insulin glargine precursor
Method, comprises the following steps：
Insulin glargine precursor is dissolved in denaturant solution, solution I is obtained；Add reducing agent to be reduced, adjust
It is 9.5~11.5 to save pH value, and the temperature for controlling reaction system I is 35 DEG C~45 DEG C, 30~60min is reacted, after being denatured
Insulin glargine precursor solution；Insulin glargine precursor solution after denaturation is added in dilution buffer, solution II is obtained；To
Protein folding additive is added in solution II, regulation pH value is 9.5~11.5；Then it is continually fed into sky into obtained solution
Gas, the temperature for controlling reaction system II is 0 DEG C~20 DEG C, is reacted 2~40 hours, obtains insulin glargine renaturation solution.
Described insulin glargine precursor is to be expressed to obtain with inclusion bodies by recombinant DNA technology by Escherichia coli,
Specifically the carry out structure of modification of actrapid monotard is formed using gene recombination technology, is 20A-Gly21-C peptides -30B-Arg31-
Arg32, i.e., replaced the 21st amino acids of actrapid monotard's A chains by glycine, while introducing two after the amino acids of B chains the 30th
Individual arginine, then expressed with being transferred to after C peptides connection A chains and B chains in Escherichia coli.
The concentration of insulin glargine precursor in described solution I is preferably 17~28g/L.
Described denaturant is urea or guanidine hydrochloride.
Described denaturant solution is preferably 6~8M urea liquids or guanidine hydrochloride solution, and pH value is 9.5~11.5；Enter one
Walk preferably 6~8M, the urea liquid of pH9.5~11.5.
Described reducing agent is selected from dithiothreitol (DTT)（DTT）One or both of with cysteine hydrochloride.
The consumption of described reducing agent is to compare 1 by reducing agent and insulin glargine precursor mass:2~1:4 calculate.
Described dilution buffer is selected from trishydroxymethylaminomethane（Tris）, glycine, in ammonium hydrogen carbonate and phosphate
A kind of buffer solution prepared.
Described dilution buffer be preferably 10~50mM, the Tris-HCl buffer solutions of pH9.5~11.5 or 10~50mM,
The glycine buffer of pH9.5~11.5.
The final concentration of insulin glargine precursor after being denatured in described solution II is preferably 0.8~2.0g/L, more preferably
For 1.0~1.4g/L.
Described protein folding additive is at least one of polyethylene glycol, glycerine and metal ion.
Described polyethylene glycol is PEG4000 or PEG6000.
Described metal ion is Ca2+、Mg2+、Cu2+And Fe3+In one kind.
Described protein folding additive is preferably that, using the volume of described solution II as denominator, concentration is 0.5~2g/
L polyethylene glycol, the glycerine of percent by volume 5%~20% and 5~50 μM of metal ion composition, more preferably concentration is
0.5~2g/L Macrogol 4000, the glycerine of percent by volume 5%~20% and 5~20 μM of Cu2+Composition.
Described Cu2+By CuSO4Or CuCl2There is provided.
It is described be passed through air be preferably by air by every liter of solution with 2~100cm3/ H speed is led into solution
Enter.
Described reaction system II reaction condition is preferably 4~15 DEG C and reacted 12~25 hours.
The refolding method of described insulin glargine precursor, more specifically comprises the following steps：
（1）The denaturation method of insulin glargine：By insulin glargine precursor be dissolved in 6~8M, pH9.5~11.5 denaturation
In agent solution, solution I is obtained；Reducing agent is added, regulation pH value is 9.5~11.5, wherein reducing agent and insulin glargine precursor
In mass ratio 1:2~1:4 proportionings, the temperature for controlling reaction system is 35 DEG C~45 DEG C, 30~60min is reacted, after being denatured
Insulin glargine precursor solution；
（2）The refolding method of insulin glargine：Insulin glargine precursor solution after denaturation is added in dilution buffer,
Obtain solution II；Polyethylene glycol, glycerine and metal ion are added into solution II, wherein, on the basis of the volume of solution II, gather
The concentration of ethylene glycol is 0.5~2g/L, and the concentration of glycerine is that percent by volume 5%~20%, the concentration of metal ion are 5~50 μ
M, regulation pH value is 9.5~11.5；Then air is continually fed into resulting solution, the temperature 0 DEG C~20 of reaction system is controlled
DEG C, react 2~40 hours, obtain insulin glargine renaturation solution；
Step（1）Described in insulin glargine precursor be by Escherichia coli by recombinant DNA technology with inclusion bodies
Expression obtain, specifically the carry out structure of modification of actrapid monotard is formed using gene recombination technology, be 20A-Gly21-C peptides-
30B-Arg31-Arg32, i.e., replaced the 21st amino acids of actrapid monotard's A chains by glycine, while in the bit amino of B chains the 30th
Two arginine are introduced after acid, then are expressed with being transferred to after C peptides connection A chains and B chains in Escherichia coli；It is preferred that by sweet smart pancreas islet
Plain precursor is dissolved in 6~8M denaturant solution by 17~28g/L concentration；
Step（1）Described in denaturant be preferably urea or guanidine hydrochloride；
Step（1）Described in reducing agent be preferably one or both of DTT and cysteine hydrochloride；
Step（2）Described in solution II in be denatured after the final concentration of insulin glargine precursor be preferably 0.8~2.0g/
L, more preferably 1.0~1.4g/L；
Step（2）Described in dilution buffer be selected from trishydroxymethylaminomethane（Tris）, glycine, ammonium hydrogen carbonate and
The buffer solution that a kind of preparation in phosphate is obtained；
The dilution buffer be preferably 10~50mM, the Tris-HCl buffer solutions of pH9.5~11.5 or 10~50mM,
The glycine buffer of pH9.5~11.5；
Step（2）Described in one kind in PEG4000 and PEG6000 of polyethylene glycol；Preferably polyethylene glycol
4000；
Step（2）Described in metal ion be Ca2+、Mg2+、Cu2+And Fe3+In one kind；Preferably by CuSO4Or
CuCl2The Cu of offer2+；Concentration is preferably 5~20 μM；
Step（2）Described in be passed through air be preferably by air by every liter of solution with 2~100cm3/ H speed to
It is passed through in solution；
Step（2）Described in the reaction temperature of reaction system be preferably 4~15 DEG C；
Step（2）Described in reaction time of reaction system be preferably 12~25 hours.
The additional proportion of reducing agent in the present invention is vital in insulin glargine precursor renaturation process.Sweet smart pancreas islet
Plain precursor contains three pairs of disulfide bond, and during Bacillus coli expression formation inclusion body, the disulfide bond mispairing in interchain and chain is led
Cause albumen inactive and handled, it is necessary to add SH-group reductant to reduce these disulfide bond, the additional proportion of reducing agent needs
In view of follow-up renaturation process.When adding the reducing agent of excessive concentrations, mistake in opening inclusion body can not only be reduced and matched somebody with somebody
To disulfide bond, moreover it is possible to reduce the disulfide bond correctly matched in inclusion body, difficulty caused to follow-up renaturation reaction, and low concentration
Reducing agent due to can not thoroughly reduce in inclusion body the disulfide bond of mistake pairing, can equally directly affect mesh in renaturation reaction
Albumen correctly fold and disulfide bond correctly match.The mass ratio of of the invention preferably reducing agent and insulin glargine precursor is preferably
1:3 add reducing agent.
The addition of protein folding additives polyethylene glycol, glycerine and metal ion is to insulin glargine precursor in the present invention
The efficiency of renaturation improves very big.The dilution oxidizing and refolding system of insulin glargine precursor, is to be used as oxidation using the oxygen in air
Agent.Protein folding additive can reduce aggregation, promote to fold, such as polyethylene glycol can protect molten spheroid, increase viscosity, sweet
Oil has preferential aquation to protein, and metal ion can be with the air oxidation speed of reducing agent residue in catalytic proteins
Rate.The addition of protein folding additive, can accelerate to be formed the disulfide bond correctly matched, so as to effectively increase correct folding
Destination protein content, while also substantially reducing the renaturation reaction time of inclusion body.The present invention is preferably the poly- second of 0.5~2g/L
Glycol 4000, metal ion solution is 5~20 μM of CuSO4Or CuCl2。
The present invention has the following advantages and effect relative to prior art：
（1）Insulin glargine precursor refolding method of the present invention, the accessory substance of disulfide bond mispairing is few, the renaturation of gained
Liquid is more beneficial for follow-up purifying process.
（2）The present invention is shortened anti-using protein folding additives polyethylene glycol, glycerine and metal ion auxiliary renaturation
Between seasonable, the protein content correctly folded is improved, annealing efficiency is greatly improved to 51%~62%.
（3）Refolding method of the present invention, using dilution refolding, using air as oxidant, greatly reduces life
Cost is produced, and is adapted to industrialized production application.
Brief description of the drawings
Sample HPLC collection of illustrative plates figures after the renaturation of Fig. 1 embodiments 1.
Sample HPLC collection of illustrative plates figures after the renaturation of Fig. 2 embodiments 2.
Sample HPLC collection of illustrative plates figures after the renaturation of Fig. 3 embodiments 3.
Sample HPLC collection of illustrative plates figures after the renaturation of Fig. 4 embodiments 4.
Embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
The embodiment of the present invention is used to detect annealing efficiency in renaturation process（%）Formula it is as follows：
Annealing efficiency（%）=（Insulin glargine precursor before the insulin glargine precursor mass/renaturation correctly folded after renaturation
Quality）×100%
Experiment material：
Insulin glargine precursor：Zhuhai United Laboratories Ltd provides, lot number F18121101；Specially by U.S.
State patent US6100376（A21,B30,MODIFIED INSULIN DERIVATIVES HAVING AN ALTERED ACTION
PROFILE）It is prepared by middle embodiment 6.
Urea derives from Guangzhou West Gansu Province Chemical Co., Ltd.；Cysteine hydrochloride has from the long-range great first share in Wuhan
Limit company；Trishydroxymethylaminomethane derives from Beijing Ke Ao Science and Technology Ltd.s；Macrogol 4000 is from Hunan that health
Pharmacy stock Co., Ltd；Glycerol source is in Hu'nan Erkang Pharmaceutical Co., Ltd.；Copper sulphate derives from Guangzhou West Gansu Province chemical industry
Co., Ltd；Copper chloride derives from Guangzhou West Gansu Province Chemical Co., Ltd.；Hydrochloric acid derives from Hua Chengda Chemical Co., Ltd.s of Zhuhai City；
Sodium hydroxide derives from Dong Jiang chemical reagent Co., Ltd.
Aeration equipment：E+H mass air flow sensor, compressed air.
The renaturation of the insulin glargine precursor of embodiment 1
At room temperature, 10 grams of insulin glargine precursors are dissolved in 588mL6M, pH9.5 urea liquid, make sweet smart pancreas islet
The protein concentration of plain precursor is about 17g/L；5g DTT are added, regulation pH value is 9.5, the temperature for controlling reaction system is 35
DEG C, react 30min.
In the Tris-HCl buffer solutions that insulin glargine precursor solution after denaturation is added to 10mM, pH9.5, Tris- is used
HCl buffer solutions are settled to 10L, make insulin glargine precursor protein concentration be 1.0g/L, be subsequently added into 5g PEG4000,
500mL glycerine, 8mg CuSO4, regulation pH value is 9.5, with 20cm3/ H speed is continually fed into air into renaturation solution, and control is anti-
The temperature for answering system is 4 DEG C, and reaction stops the reaction after 12 hours.Take the μ L of renaturation solution 20 to analyze through HPLC to detect, after renaturation
Sample HPLC collection of illustrative plates is shown in Fig. 1, and the retention time of the insulin glargine precursor HPLC collection of illustrative plates of correct refolding is 19.98min, root
Calculated according to the areas of peak normalization method of insulin glargine precursor before the insulin glargine that correct refolding is actually obtained after renaturation
The amount of body is 6.1g, and the yield of insulin glargine precursor renaturation is computed about 61%.
The renaturation of the insulin glargine precursor of embodiment 2
At room temperature, 10 grams of insulin glargine precursors are dissolved in 357mL8M, pH11.5 urea liquid, make sweet smart pancreas
The protein concentration of island element precursor is about 28g/L, adds 2.5g DTT, regulation pH value is 11.5, and the temperature for controlling reaction system is
45 DEG C, react 60min.
In the Tris-HCl buffer solutions that insulin glargine precursor solution after denaturation is added to 50mM, pH11.5, Tris- is used
HCl buffer solutions are settled to 7.14L, and the protein concentration for making insulin glargine precursor is 1.4g/L, is subsequently added into 14.28g
PEG4000,1428mL glycerine, 22.85mg CuSO4, regulation pH value is 11.5, with 714cm3/ H speed continues into renaturation solution
Air is passed through, 15 DEG C of the temperature of reaction system is controlled, reaction stops the reaction after 25 hours.The μ L of renaturation solution 20 are taken to be analyzed through HPLC
Sample HPLC collection of illustrative plates after detection, renaturation is shown in Fig. 2, the retention time of the insulin glargine precursor HPLC collection of illustrative plates of correct refolding
For 20.71min, correct refolding is actually obtained after calculating renaturation according to the areas of peak normalization method of insulin glargine precursor
The amount of insulin glargine precursor is 5.14g, and the yield of insulin glargine precursor renaturation is computed about 51.40%.
The renaturation of the insulin glargine precursor of embodiment 3
At room temperature, 10 grams of insulin glargine precursors are dissolved in 588mL6M, pH9.5 urea liquid, make sweet smart pancreas islet
The protein concentration of plain precursor is about 17g/L, adds 5g cysteine hydrochlorides, and regulation pH value is 9.5, controls the temperature of reaction system
Spend for 35 DEG C, react 30min.
In the glycine dilution buffer that insulin glargine precursor solution after denaturation is added to 10mM, pH9.5, sweet ammonia is used
Sour dilution buffer is settled to 10L, make insulin glargine precursor protein concentration be 1.0g/L, be subsequently added into 5g PEG4000,
500mL glycerine, 6.75mg CuCl2, regulation pH value is 9.5, with 20cm3/ H speed is continually fed into air into renaturation solution, control
4 DEG C of the temperature of reaction system processed, reaction stops the reaction after 12 hours.Take the μ L of renaturation solution 20 to analyze through HPLC to detect, after renaturation
Sample HPLC collection of illustrative plates see Fig. 3, the retention time of the insulin glargine precursor HPLC collection of illustrative plates of correct refolding is 19.94min,
The insulin glargine that correct refolding is actually obtained after renaturation is calculated according to the areas of peak normalization method of insulin glargine precursor
The amount of precursor is 6.2g, and the yield of insulin glargine precursor renaturation is computed about 62%.
The renaturation of the insulin glargine precursor of embodiment 4
At room temperature, 10 grams of insulin glargine precursors are dissolved in 357mL8M, pH11.5 urea liquid, make sweet smart pancreas
The protein concentration of island element precursor is about 28g/L, adds 2.5g cysteine hydrochlorides, and regulation pH value is 11.5, controls reactant
The temperature of system is 45 DEG C, reacts 60min.
In the glycine dilution buffer that insulin glargine precursor solution after denaturation is added to 50mM, pH11.5, with sweet
Propylhomoserin dilution buffer is settled to 7.14L, and the protein concentration for making insulin glargine precursor is 1.4g/L, is subsequently added into 14.28g
The CuCl of PEG4000,1428mL glycerine, 19.28mg2, regulation pH value is 11.5, with 714cm3/ H speed is held into renaturation solution
It is continuous to be passed through air, 15 DEG C of the temperature of reaction system is controlled, reaction stops the reaction after 25 hours.The μ L of renaturation solution 20 are taken through HPLC points
Sample HPLC collection of illustrative plates after analysis detection, renaturation is shown in Fig. 4, during the reservation of the insulin glargine precursor HPLC collection of illustrative plates of correct refolding
Between be 20.75min, according to the areas of peak normalization method of insulin glargine precursor calculate renaturation after actually obtain correct refolding
The amount of insulin glargine precursor be 5.35g, the yield of insulin glargine precursor renaturation is computed about 53.5%.
Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention
Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (10)
1. a kind of refolding method of insulin glargine precursor, it is characterised in that comprise the following steps：Insulin glargine precursor is molten
Solution obtains solution I in denaturant solution；Add reducing agent to be reduced, regulation pH value is 9.5~11.5, is reacted
System I；The temperature for controlling reaction system I is 35 DEG C~45 DEG C, is reacted before 30~60min, the insulin glargine after being denatured
Liquid solution；Insulin glargine precursor solution after denaturation is added in dilution buffer, solution II is obtained；Added into solution II
Protein folding additive, regulation pH value is 9.5~11.5；Then air is continually fed into obtained solution, is reacted
System II；The temperature for controlling reaction system II is 0 DEG C~20 DEG C, is reacted 2~40 hours, obtains insulin glargine renaturation solution；
Described reducing agent is one or both of dithiothreitol (DTT) and cysteine hydrochloride；
The consumption of described reducing agent is to compare 1 by reducing agent and insulin glargine precursor mass:2~1:4 calculate.
2. the refolding method of insulin glargine precursor according to claim 1, it is characterised in that：
The concentration of insulin glargine precursor in described solution I is 17~28g/L；
Final concentration of 0.8~2.0g/L of insulin glargine precursor after being denatured in described solution II.
3. the refolding method of insulin glargine precursor according to claim 2, it is characterised in that：Become in described solution II
Final concentration of 1.0~1.4g/L of insulin glargine precursor after property.
4. the refolding method of insulin glargine precursor according to claim 1, it is characterised in that：
Described denaturant is urea or guanidine hydrochloride；
Described dilution buffer is that one kind in trishydroxymethylaminomethane, glycine, ammonium hydrogen carbonate and phosphate is prepared into
The buffer solution arrived；
Described protein folding additive is at least one of polyethylene glycol, glycerine and metal ion.
5. the refolding method of insulin glargine precursor according to claim 4, it is characterised in that：
Described denaturant solution is 6~8M, pH 9.5~11.5 urea liquid or guanidine hydrochloride solution；
Described dilution buffer is 10~50mM, pH 9.5~11.5 Tris-HCl buffer solutions or 10~50mM, pH 9.5
~11.5 glycine buffer.
6. the refolding method of insulin glargine precursor according to claim 4, it is characterised in that：
Described polyethylene glycol is PEG4000 or PEG6000；
Described metal ion is Ca2+、Mg2+、Cu2+And Fe3+In one kind；
Described protein folding additive is by final concentration of 0.5~2g/L polyethylene glycol, percent by volume 5%~20%
Glycerine and 5~50 μM of metal ion composition.
7. the refolding method of insulin glargine precursor according to claim 6, it is characterised in that：Described protein folding
Additive is the PEG4000, the glycerine of percent by volume 5%~20% and 5~20 μM of Cu by final concentration of 0.5~2g/L2+Group
Into.
8. the refolding method of insulin glargine precursor according to claim 1, it is characterised in that：The described air that is passed through is
Air is pressed in every liter of solution with 2~100cm3/ H speed is passed through into solution；
Described reaction system II reaction condition is 4~15 DEG C and reacted 12~25 hours.
9. the refolding method of the insulin glargine precursor according to any one of claim 1~8, it is characterised in that including as follows
Step：
（1）The denaturation method of insulin glargine：Insulin glargine precursor is dissolved in 6~8M, pH 9.5~11.5 denaturant
In solution, solution I is obtained；Reducing agent is added, regulation pH value is 9.5~11.5, obtains reaction system I；Wherein reducing agent with it is sweet
Smart insulin precurosor in mass ratio 1:2~1:4 proportionings, the temperature for controlling reaction system I is 35 DEG C~45 DEG C, reaction 30~
60min, the insulin glargine precursor solution after being denatured；
（2）The refolding method of insulin glargine：Insulin glargine precursor solution after denaturation is added in dilution buffer, obtained
Solution II；Polyethylene glycol, glycerine and metal ion are added into solution II, wherein, final concentration of 0.5~2g/ of polyethylene glycol
L, the final concentration of percent by volume 5%~20% of glycerine, final concentration of 5~50 μM of metal ion, regulation pH value is 9.5~
11.5；Then air is continually fed into resulting solution, reaction system II is obtained；Control reaction system II temperature 0 DEG C~20
DEG C, react 2~40 hours, obtain insulin glargine renaturation solution.
10. the refolding method of insulin glargine precursor according to claim 9, it is characterised in that：
Step（1）Described in solution I in insulin glargine precursor concentration be 17~28g/L；
Step（1）Described in denaturant be urea or guanidine hydrochloride；
Step（1）Described in reducing agent be one or both of DTT and cysteine hydrochloride；
Step（2）Described in solution II in be denatured after insulin glargine precursor final concentration of 1.0~1.4g/L；
The dilution buffer be 10~50mM, pH 9.5~11.5 Tris-HCl buffer solutions or 10~50mM, pH 9.5~
11.5 glycine buffer；
Described polyethylene glycol is PEG4000；
Step（2）Described in metal ion be Cu2+, concentration is 5~20 μM；
Step（2）Described in be passed through air be by air by every liter of solution with 2~100cm3/ H speed is led into solution
Enter；
Step（2）Described in reaction system II reaction temperature be 4~15 DEG C；
Step（2）Described in reaction system II reaction time be 12~25 hours.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US4511502A (en) * | 1982-12-22 | 1985-04-16 | Genentech, Inc. | Purification and activity assurance of precipitated heterologous proteins |
| US4512922A (en) * | 1982-12-22 | 1985-04-23 | Genentech, Inc. | Purification and activity assurance of precipitated heterologous proteins |
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| DE19735711C2 (en) * | 1997-08-18 | 2001-04-26 | Aventis Pharma Gmbh | Process for the preparation of a precursor to insulin or insulin derivatives with correctly linked cystine bridges |
| CN1332027C (en) * | 2005-11-01 | 2007-08-15 | 武汉大学 | Preparation method of recombination buman tPA |
| AU2007272412B2 (en) * | 2006-07-14 | 2013-11-07 | Genentech, Inc. | Refolding of recombinant proteins |
| WO2009133529A2 (en) * | 2008-04-30 | 2009-11-05 | Wockhardt Research Centre | Processes for refolding of insulin |
| CN101298610A (en) * | 2008-06-06 | 2008-11-05 | 浙江大学 | Method for assisting lysozyme in vitro refolding by means of linear poly N-isopropyl acrylamide |
| UA91281C2 (en) * | 2008-11-26 | 2010-07-12 | Общество С Ограниченной Ответственностью «Мако» | Method for producing of recombinant human insulin |
| CA2789615A1 (en) * | 2010-03-17 | 2011-09-22 | Biogenerix Gmbh | Method for obtaining biologically active recombinant human g-csf |
| WO2012115638A1 (en) * | 2011-02-23 | 2012-08-30 | Elona Biotechnologies | Glargine proinsulin compositions and methods of producing glargine insulin analogs therefrom |
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| US4511502A (en) * | 1982-12-22 | 1985-04-16 | Genentech, Inc. | Purification and activity assurance of precipitated heterologous proteins |
| US4512922A (en) * | 1982-12-22 | 1985-04-23 | Genentech, Inc. | Purification and activity assurance of precipitated heterologous proteins |
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Application publication date: 20140402 Assignee: Zhuhai United Biopharmaceutical Co.,Ltd. Assignor: ZHUHAI UNITED LABORATORIES Co.,Ltd. Contract record no.: X2024980001686 Denomination of invention: A refolding method for proinsulin glargine Granted publication date: 20171013 License type: Common License Record date: 20240131 Application publication date: 20140402 Assignee: Federal Biotechnology (Zhuhai Hengqin) Limited Co. Assignor: ZHUHAI UNITED LABORATORIES Co.,Ltd. Contract record no.: X2024980001558 Denomination of invention: A refolding method for proinsulin glargine Granted publication date: 20171013 License type: Common License Record date: 20240129 |