CA2008246A1 - Process for renaturing incorrect recombinants of insulin precursor - Google Patents
Process for renaturing incorrect recombinants of insulin precursorInfo
- Publication number
- CA2008246A1 CA2008246A1 CA002008246A CA2008246A CA2008246A1 CA 2008246 A1 CA2008246 A1 CA 2008246A1 CA 002008246 A CA002008246 A CA 002008246A CA 2008246 A CA2008246 A CA 2008246A CA 2008246 A1 CA2008246 A1 CA 2008246A1
- Authority
- CA
- Canada
- Prior art keywords
- insulin
- incorrect
- recombinants
- redox system
- organic
- 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.)
- Abandoned
Links
- 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 title claims abstract description 72
- 108090001061 Insulin Proteins 0.000 title claims abstract description 34
- 102000004877 Insulin Human genes 0.000 title claims abstract description 33
- 229940125396 insulin Drugs 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 29
- 239000002243 precursor Substances 0.000 title claims abstract description 19
- 238000006243 chemical reaction Methods 0.000 claims abstract description 23
- LSDPWZHWYPCBBB-UHFFFAOYSA-N Methanethiol Chemical compound SC LSDPWZHWYPCBBB-UHFFFAOYSA-N 0.000 claims abstract description 15
- 150000002894 organic compounds Chemical class 0.000 claims abstract description 10
- 239000012736 aqueous medium Substances 0.000 claims abstract description 5
- 235000001014 amino acid Nutrition 0.000 claims description 30
- 150000001413 amino acids Chemical class 0.000 claims description 26
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 22
- 150000001875 compounds Chemical class 0.000 claims description 21
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 21
- 239000000047 product Substances 0.000 claims description 19
- 235000010323 ascorbic acid Nutrition 0.000 claims description 10
- 239000011668 ascorbic acid Substances 0.000 claims description 10
- 229960005070 ascorbic acid Drugs 0.000 claims description 10
- YCIMNLLNPGFGHC-UHFFFAOYSA-N catechol Chemical compound OC1=CC=CC=C1O YCIMNLLNPGFGHC-UHFFFAOYSA-N 0.000 claims description 10
- QIGBRXMKCJKVMJ-UHFFFAOYSA-N Hydroquinone Chemical compound OC1=CC=C(O)C=C1 QIGBRXMKCJKVMJ-UHFFFAOYSA-N 0.000 claims description 9
- 101000976075 Homo sapiens Insulin Proteins 0.000 claims description 8
- 108010005991 Pork Regular Insulin Proteins 0.000 claims description 8
- PBGKTOXHQIOBKM-FHFVDXKLSA-N insulin (human) 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)=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)C1=CN=CN1 PBGKTOXHQIOBKM-FHFVDXKLSA-N 0.000 claims description 8
- AZQWKYJCGOJGHM-UHFFFAOYSA-N 1,4-benzoquinone Chemical compound O=C1C=CC(=O)C=C1 AZQWKYJCGOJGHM-UHFFFAOYSA-N 0.000 claims description 7
- SBJKKFFYIZUCET-JLAZNSOCSA-N Dehydro-L-ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(=O)C1=O SBJKKFFYIZUCET-JLAZNSOCSA-N 0.000 claims description 7
- SBJKKFFYIZUCET-UHFFFAOYSA-N Dehydroascorbic acid Natural products OCC(O)C1OC(=O)C(=O)C1=O SBJKKFFYIZUCET-UHFFFAOYSA-N 0.000 claims description 7
- 235000020960 dehydroascorbic acid Nutrition 0.000 claims description 7
- 239000011615 dehydroascorbic acid Substances 0.000 claims description 7
- WOAHJDHKFWSLKE-UHFFFAOYSA-N 1,2-benzoquinone Chemical group O=C1C=CC=CC1=O WOAHJDHKFWSLKE-UHFFFAOYSA-N 0.000 claims description 6
- 238000005215 recombination Methods 0.000 claims description 6
- 230000006798 recombination Effects 0.000 claims description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 5
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 claims description 5
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 5
- 101001011741 Bos taurus Insulin Proteins 0.000 claims description 4
- 125000003118 aryl group Chemical group 0.000 claims description 4
- IXIBAKNTJSCKJM-BUBXBXGNSA-N bovine insulin 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)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)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(C)C)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 IXIBAKNTJSCKJM-BUBXBXGNSA-N 0.000 claims description 4
- 239000006227 byproduct Substances 0.000 claims description 4
- 235000018417 cysteine Nutrition 0.000 claims description 4
- 239000004202 carbamide Substances 0.000 claims description 3
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 3
- 125000006239 protecting group Chemical group 0.000 claims description 2
- 239000012431 aqueous reaction media Substances 0.000 claims 1
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 10
- 230000000875 corresponding effect Effects 0.000 description 7
- 108010075254 C-Peptide Proteins 0.000 description 6
- 239000000203 mixture Substances 0.000 description 5
- 238000001556 precipitation Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- VOUAQYXWVJDEQY-QENPJCQMSA-N 33017-11-7 Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)NCC(=O)NCC(=O)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N1[C@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O)CCC1 VOUAQYXWVJDEQY-QENPJCQMSA-N 0.000 description 4
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 4
- 108010076181 Proinsulin Proteins 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 239000004471 Glycine Substances 0.000 description 3
- 108020001507 fusion proteins Proteins 0.000 description 3
- 102000037865 fusion proteins Human genes 0.000 description 3
- 238000010353 genetic engineering Methods 0.000 description 3
- 108010066381 preproinsulin Proteins 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- CRTGSPPMTACQBL-UHFFFAOYSA-N 2,3-dihydroxycyclopent-2-en-1-one Chemical compound OC1=C(O)C(=O)CC1 CRTGSPPMTACQBL-UHFFFAOYSA-N 0.000 description 2
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- SMYXEYRYCLIPIL-ZLUOBGJFSA-N Cys-Cys-Cys Chemical compound SC[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(O)=O SMYXEYRYCLIPIL-ZLUOBGJFSA-N 0.000 description 2
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical class S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 2
- PCLIMKBDDGJMGD-UHFFFAOYSA-N N-bromosuccinimide Chemical compound BrN1C(=O)CCC1=O PCLIMKBDDGJMGD-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- -1 for example Chemical group 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 108010013359 miniproinsulin Proteins 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 229910052717 sulfur Inorganic materials 0.000 description 2
- 239000011593 sulfur Substances 0.000 description 2
- CWERGRDVMFNCDR-UHFFFAOYSA-N thioglycolic acid Chemical compound OC(=O)CS CWERGRDVMFNCDR-UHFFFAOYSA-N 0.000 description 2
- CNHDIAIOKMXOLK-UHFFFAOYSA-N toluquinol Chemical compound CC1=CC(O)=CC=C1O CNHDIAIOKMXOLK-UHFFFAOYSA-N 0.000 description 2
- 125000004178 (C1-C4) alkyl group Chemical group 0.000 description 1
- JVDYMIODNJPGIB-UHFFFAOYSA-N 2,3-dihydroxy-4-methylcyclopent-2-en-1-one Chemical compound CC1CC(=O)C(O)=C1O JVDYMIODNJPGIB-UHFFFAOYSA-N 0.000 description 1
- PCFMUWBCZZUMRX-UHFFFAOYSA-N 9,10-Dihydroxyanthracene Chemical compound C1=CC=C2C(O)=C(C=CC=C3)C3=C(O)C2=C1 PCFMUWBCZZUMRX-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 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
- 241000588724 Escherichia coli Species 0.000 description 1
- NVXLIZQNSVLKPO-UHFFFAOYSA-N Glucosereductone Chemical compound O=CC(O)C=O NVXLIZQNSVLKPO-UHFFFAOYSA-N 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-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
- 229930192627 Naphthoquinone Natural products 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100028703 Protein maestro Human genes 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 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
- 229930003268 Vitamin C Natural products 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 150000001491 aromatic compounds Chemical class 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 239000012928 buffer substance Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- ATDGTVJJHBUTRL-UHFFFAOYSA-N cyanogen bromide Chemical compound BrC#N ATDGTVJJHBUTRL-UHFFFAOYSA-N 0.000 description 1
- 150000001945 cysteines Chemical class 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- VHJLVAABSRFDPM-ZXZARUISSA-N dithioerythritol Chemical compound SC[C@H](O)[C@H](O)CS VHJLVAABSRFDPM-ZXZARUISSA-N 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 239000001307 helium Substances 0.000 description 1
- 229910052734 helium Inorganic materials 0.000 description 1
- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- NWVVVBRKAWDGAB-UHFFFAOYSA-N hydroquinone methyl ether Natural products COC1=CC=C(O)C=C1 NWVVVBRKAWDGAB-UHFFFAOYSA-N 0.000 description 1
- 125000000687 hydroquinonyl group Chemical group C1(O)=C(C=C(O)C=C1)* 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 125000002768 hydroxyalkyl group Chemical group 0.000 description 1
- 239000004026 insulin derivative Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- PCILLCXFKWDRMK-UHFFFAOYSA-N naphthalene-1,4-diol Chemical compound C1=CC=C2C(O)=CC=C(O)C2=C1 PCILLCXFKWDRMK-UHFFFAOYSA-N 0.000 description 1
- 150000002791 naphthoquinones Chemical class 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 125000000962 organic group Chemical group 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001451 polypropylene glycol Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 150000004053 quinones Chemical class 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 230000002311 subsequent effect Effects 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Toxicology (AREA)
- Endocrinology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Diabetes (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
Abstract of the disclosures Process for renaturing incorrect recombinants of insulin precursors "Incorrect" recombinants of insulin precursors are renatured in an aqueous medium using excess mercaptan in the presence of an organic redox system or at least one organic compound which forms such an organic redox system under the reaction conditions, that is to say converted into the "correct" recombinant in a single reaction step.
The "correct" recombinants can be converted into the corresponding insulin enzymatically or chemically by known techniques.
The "correct" recombinants can be converted into the corresponding insulin enzymatically or chemically by known techniques.
Description
fi HOECHST AKTIENGESELLSCHAET HOE 8~/F 020 Dr.ME/rh Description Process for renaturing incorrect recombinants of insulin precursors Insulin is a molecule which consists of 2 polypeptide chains linked to one another via disulfide bridges. The A chain consists of 21 amino acids and the B chain of 30 amino acids. These two chains are linked to one another in the precursor molecule, the proinsulin, by a peptide, the C-peptide. The C-peptide in human proinsulin consists of 35 amino acids. In the context of the maturation process of the hormone, the C-peptide is split off by specific proteases and the proinsulin is thus converted into insulin (Davidson et al., Nature 333, 93-96, 1988).
In addition to the naturally occurring C-peptides, a large number of ~oining possibilities between the A ohain and B chain are described in the literature (Yanaihara et al., Diabetes 27, 149-160 (1978), Busse et al., Bio-chemistry 15, 1649-1657 (1971), and Geiger et al., Bio-chem. Biophys. Res. Com. 55, 60-66 (1973)).
In the context of genetic engineering, it is now possible to prepare insulin from microorganisms modified by genetic engineering. If E. coli is used as the micro-organism, the product is frequently expressed as fusion protein, that is to say the product is coupled with a protein endogenous to the bacteria, for example with ~-galactosidase. This fusion protein precipitates out in the cell and is in this way protected from proteolytic degradation. After breakdown of the cell, the fusion protein content i8 split off chemically or enzymatically and the 6 cysteines of the insulin precursor are con-verted into their S-sulfonates (-S-S03- ) by means of oxidative sulfitolysis. In a subsequent ("renaturing or recombination~) step, natural preproinsulin must be produced from this so-called preproinsulin S-sulfonate by formation of the 3 correct disulfide bridges - that is to ~ L~ 6 say -S-S- bridges from A6 to All, from A7 to B7 and from A20 to Bl9 in the corresponding insulin peptide sequen-ces.
According to the process described in EP-B-0,037,255, this step is carried out, for example, by reaction of the starting S-sulfonate with a mercaptan in an amount which results in 1 to 5 SH radicals per SS03- radical in an aqueous medium at a pH of 7 to 11.5 and an S-sulfonate concentration of up to 10 mg per ml of aqueous medium, preferably in the absence of an oxidizing agent.
Yields of in some cases more than 80% are said to be obtained here.
In addition to the (desired) renaturing products with correct disulfide bridges, more or less substantial amounts of ~undesired) "incorrect" recombinants, that is to say insulin products with disulfide bridges which are only partly correct or not correct at all and also with intermolecular disulfide bridges, are always also formed - depending on the reaction conditions and in particular the concentration circumstances - in this and practically all other known renaturing or recombination processes in which insulin precursors with opened disulfide bridges are converted into products with the correspondingly closed disulfide bridges.
When the insulin precursor products obtained by the known renaturing processes are worked up to insulin without the "incorrect" recombinants being removed - this working up being effected by known techniques (chemically or enzymatically) - no (natural) insulin is formed from the "incorrect" recombinants.
It is therefore advantageous or necessary to remove the ~incorrect" recombinants from the renaturing products with the correct disulfide bridges before working up the corresponding renaturing products to give insulin. This ~0(~ 4fi can be effected, for example, by known chromatographic processes. In another particularly advantageous process, the removal is effected by adjusting the reaction mixture to pH 4 to 6 - preferably in the presence of a small amount of a physiologically acceptable surface-active substance - the ~correct recombinants remaining virtu-ally completely in solution and the "incorrect" recom-binants being precipitated (c.f. DE-A-35 01 641).
The "incorreck" recombinants removed are then advantage-ously converted back by sulfitolysis into the correspond-ing S-sulfonate, which is subjected to renewed folding, it often being necessary to remove by-products formed during the sulfitolysis by chromatography before the renaturing. The ~incorrect recombinants can in this way largely be converted back into "usable" product.
Sulfitolysis of the "incorrect" recombinants with subse-quent chromatography and renewed folding of course means a not inconsiderable expenditure.
In the efforts to avoid or at least reduce this expendi-ture, it has now been found that this is possible byreacting the "incorrect" recombinants of insulin precur-sors with excess mercaptan in an aqueous medium in the presence of an organic redox system or at least one organic compound which forms such an organic redox system under the reaction conditions.
The "incorrect" recombinants can in this way be converted in high yields directly into the "correct" renaturing products or "correct" recombinants without sulfitolysis, which represents a considerable advantage compared with the prior art.
There is also no prior art at all which would have made obvious in any manner bypassing of sulfitolysis with the subsequent folding stage merely by reaction with excess mercaptan and the addition of an organic redox system.
~OC~
Possible incorrect recombinants of insulin precursors for the process accordin~ to the invention are preferably the products which are formed as by-products on recom-bination of insulin precursors with opened -S-S- bridges of the following formula I:
(A-1) Gly-NH -- X
I
(A-6) Cys-S-R3 S-R3 I l (A-20) (A-21) (A-7) Cys-------Cys--------- Cys - Z - R2 I (A~ (I) (3-1) 1 l R1-HN-Phe---Cys------------------~ Cys--------------Y
~B-7) (B-19) (B-30) in which R~ = H or an amino acid or peptide radical which can be split off chemically or enzymatically, R2 = OH or an amino acid or peptide radical, preferably OH, R3 - H or a Cy~-5 protective group, preferably the -SO3- or the tert.-butyl group, X = a radical which ~oins the insulin A and B
chains, preferably an amino acid or peptide radical, Y = the radical of a genetically encodable amino acid, preferably Thr, Ala or Ser, in particular Thr, Z = the radical of a genetically encodable amino acid, preferably Asn, Gln, Asp, Glu, Gly, Ser, Thr, Ala or Met, in particular Asn, and ~l-A2Q and Bl-B29 = peptide sequences of insulin which are non-mutated or mutated by re-placement of one or more amino acids, - s -preferably the non-mutated peptide sequen-ces of human, porcine or bovine insulin, in particular of human or porcine insulin.
If R, = H in formula I, the products are products which are derived from proinsulin; if Rl = an amino acid or peptide radical which can be split off chemically or enzymatically, the products are products which are derived from preproinsulin.
Amino acid radicals which can be split off chemically are those which are split off, for example, by means of BrCN
or N-bromosuccinimide; these are, for example, methionine (Met) or tryptophan (Trp).
Amino acid radicals which can be split off enzymatically are those which can be split off, for example, by means of trypsin (such as, for example, Arq or Lys).
Peptide radicals which can be split off chemically or enzymatically are peptide radica's having at least 2 amino acids.
All the amino acids possible for Rl are preferably those of the group of naturally occurring amino acids, that is to ~ay mainly Gly, Ala, Ser, Thr, Val, Leu, Ile, Asn, Gln, Cys, Met, Tyr, Phe, Pro, Hyp, Arg, Lys, Hyl, Orn, Cit and His.
R2 is OH or - sLmilarly to Rl - likewise an amino acid or peptide radical, the meaning of OH being preferred. The amino acids (including those which form the peptide radical - consisting of at ~east 2 amino acid radicals) preferably originate - as for Rl - from the group of naturally occurring amino acids.
R3 is hydrogen or a cysteine-sulfur protective group, the -S03- or the tert.-butyl group being preferred cysteine-sulfur protective groups.
;~0~ 6 X is a radical which joins the insulin A and B chains, preferably an amino acid or peptide radical.
If X is an amino acid radical, the radical of Arg or Lys is preferred; if X is a peptide radical, the radical of a naturally occurring C-peptide - in particular the human, porcine or bovine insulin C-peptide - is preferred.
Genetically encodable amino acids - for Y - are (in each case in the L form): Gly, Ala, Ser, Thr, Val, Leu, Ile, Asp, Asn, Glu, Gln, Cys, Met, Arg, Lys, His, Tyr, Phe, Trp and Pro.
Preferred genetically encodable amino acids are Thr, Ala and Ser, in particular Thr.
Z can - like Y - also denote the radical of a genetically encodable amino acid, but in this case Asn, Gln, Asp, Glu, Gly, Ser, Thr, Ala and Met, in particular Asn, are preferred.
Al - A20 and Bl - B29 can in principle be the peptide sequences which are non-mutated or mutated by replacement of one or more amino acids and originate from all pO8-sible insulins; the mutants can be produced by known processes of genetic engineering (site directed mutagene-sis~. However, the non-mutated peptide sequences of human, porcine or bovine insulin, in particular of human or porcine insulin ~the Al - A20 and B1 - B29 sequences of human and porcine insulin are identical) are preferred.
The "incorrect" recombinants formed as by-products during recombination of insulin precursors with opened -S-S-bridges of the formula I are removed from the "correct"
recombinants in a known manner - preferably by precipita-tion at pH values of ~ to 6 in accordance with the process of the abovementioned DE-A-35 01 641 - and are X0(~46 then dissolved directly, or after prior freeze-drying, in water or an aqueous solution for use for the process according to the invention.
The concentration of the "incorrect" recombinants in the aqueous starting solution can vary within a wide range;
preferred concentrations are about 0.1 to about 100 mg, in particular about 0.1 to about 10 mg/ml, the mg values relating to the "incorrect~ recombinants as dried solid.
Suitable mercaptans for the reaction according to the invention are in principle all the possible organic compounds with SH groups; mercaptoethanol, thioglycolic acid, dithioerythritol, qlutathione and cysteine, in particular mercaptoethanol and cysteine, are preferred.
The mercaptans can be employed individually or as a mixture.
The amount of mercaptan to be employed can vary within wide limits; an excess of mercaptan corresponding to a ratio of mercaptan-SH groups~cysteine-S units (in the ~incorrect~' recombinants) of at least about 5 ifi pre-ferred. This ratio has an upper limit imposed practicallyonly by economic considerations. An upper limit of about 100 i8 advantageous.
Because of the wide range of variation of the excess mercaptan, the number of cysteine-S units in the "in-correct" recombinant employed does not have to be deter-mined completely accurately.
Preferred possible organic redox systems are pairs of compounds, one component of which is an organic compound having the structural element of the formula II
OH OH O
1~ (II) O OH OH
-- C -- C _ C --ZOG~ 6 or an aromatic o- or p-dihydroxy compound and the other component of which is an organic compound having the structural element of the formula II in oxidized form =
structural element of the formula II' O O O
5- C - C - C - (II') or is an o- or p-quinone.
The free valencies of the structural element of the formula II and II' can be satisfied by hydrogen or organic groups, such as, for example, C1-C4-alkyl groups.
However, ~he structural element can also be part of a ring having preferably 4, 5 or 6 carbon ring atoms and if appropriate also one or two hetero atoms, such as, for example, O, it being possible for the ring in turn to be substituted by groups which are inert under the reaction conditions, such as, for example, alkyl or hydroxyalkyl groups.
Examples of compounds having the structural element of the formula II are:
reductone OH OH O
H - C = C - C - H
reductic acid pH OH
HC - CH
\C' o methylreductic acid OH OH
H~ -- CH
C O
X~ 6 g ascorbic acid OH OH
(vitamin C) OH HC CH
O
The formulae here are in each case written only in one of the tautomeric forms.
All the compounds are reducing. In the oxidized form, the structural element of the formula II becomes that of the formula II'.
Possible aromatic o- and p-dihydroxy compounds are in principle all the possible aromatic compounds having two OH groups in the o- or p-position, it merely being necessary that the o- or p-quinone formation from the o-and p-dihydroxy compounds cannot be prevented by any particular substituents or the like. Examples of aromatic o- and p-dihydroxy compounds are 1,2-dihydroxybenzene = pyrocatechol, 1,4-dihdyroxybenzene = hydroquinone, methyl-hydroquinone, naphtho-1,4-hydroxyguinone and anthra-hydroquinone; on oxidation, the corresponding quinones are formed therefrom.
In the reaction according to the invention the particular organic redox systems consisting, for example, of the pairs of compounds of ascorbic acid + dehydroascorbic acid, pyrocatechol + o-quinone, hydroquinone + p-quinone, naphthohydroquinone + naphthoquinone and the like, can thus be employed in virtually any desired ratio (prefer-ably in an approximately equimolar ratio). However, it is also possible for only the particular individual com-ponen~s of these pairs of compounds - that is to say, for example, only ascorbic acid or only dehydroascorbic acid or only hydroquinone and the like - to be added, because the other particular component belonying to the redox pair of compounds ~dehydroascorbic acid or ascorbic acid or p-quinone and the like) forms in the reaction medium.
x~ >~
Preferred organic redox systems are the combinations consisting of the pairs of compounds ascorbic acid + dehydroascorbic acid, pyrocatechol + o-quinone and hydroquinone + p-quinone and preferred individual compounds which form such a redox system under the reaction conditions are the individual components of these pairs of compounds.
Ascorbic acid and/or dehydroascorbic acid are especially preferred.
The amount employed of the compound(s) which form(s) the organic redox system can vary within wide limits. The number of mol of compound(s) which fo~m(s) the organic redox system can be chosen as being between about 1/10,000 and 10,000, preferably between about 1/10 and 10, based on one gram equivalent of mercaptan (= mole-cular weight of the mercaptan employed in g/number of SH
groups in the mercaptan molecule)~
It is advantageous also to add urea to the reaction solution, concentrations corresponding to about 0.1 to 1 M ~M = molar), in particular 0.1 - 0.5 M, being pre-ferred.
The reaction according to the invention is advantageously carried out in the alkaline pH range, preferably between about 7 and 12, in particular between about 9.5 and 11.
To maintain the desired pH, it is advantageous to add a buffer substance, the nature and ionic strength of the buffer having a certain influence on the folding yield.
It is advantageous to keep the ionic strength low, a 3~ range of about 1 mM (mM = millimolar) to 1 M (M = molar), in particular one from about 5 mM to 50 mM, being pre-ferred. Buffer substances which can be used are, for example, borate buffer, carbonate buffer or glycine buffer, the latter being preferred.
XO~ ~4fi A general range of reaction temperature which may be stated is one between about 0 and 45C; a range from about 4 to 8DC is preferred.
Covering the renaturing solution with a layer of certain gases, such as, for example, oxygen, nitrogen or helium, has no noticeable influence on the renaturing yield.
The reaction time is in general between about ~ and 24 hours, preferably between about 6 and 16 hours.
The renaturing product of the reaction according to the invention is - if the "incorrect" starting recombinant originates from recombination of an insulin precursor with opened S-S bridges of the abovementioned formula I -an insulin precursor having correct disulfide bonds (~correct" recombinant) of the formula III
(A-1) Gly-NH X
I
(A-6) Cys-S-S
I ¦ (A-20) (A-21) (A-7) Cys---Cys--------- Cys - Z - R2 ¦ (A-11) l (III) S S
(B-1) l l Rl-HN-Phe---Cys--------------- Cy~------------Y
(~-7) tB-19) (B-30) in which Rl, R2, X, Y and Z have the same meaning as in formula I.
When the reaction has ended (which can be ascertained, - for example, by high performance liquid chromatography), the mixture is worked up in a known manner.
The "correctly" folded product, preferably of the formula III, can then be converted into the corresponding insulin enzymatically or chemically by known techniques.
~o~
The following example is intended to illustrate the invention in more detail. Before the (invention) example, the preparation of the starting substance is also des-cribed by way of example.
A) Preparation of the starting substance 1. Folding of "miniproinsulin"
~Miniproinsulin-S-SO3~, that is to say an insulin precursor in which the A and B chains of the insulin are linked via an arginine and the B chain is leng-thened N-terminally, is employed. The freeze-dried material (60% pure) is dissolved at a solids con-centration of 0.5 g/l in 50 mM glycine buffer, pH
10.7, which corresponds to a precursor concentration of 0.3 g/l. 630 ml of 1 M mercaptoethanol and 630 ml of 1 M ascorbic acid are added to the batch (100 1), and the mixture is then stirred slowly in a cold chamber at 8C for 16 hours. The folding yield, determined by high performance liquid chromatography against a standard, is 0.228 g/l (76% of theory).
In addition to the naturally occurring C-peptides, a large number of ~oining possibilities between the A ohain and B chain are described in the literature (Yanaihara et al., Diabetes 27, 149-160 (1978), Busse et al., Bio-chemistry 15, 1649-1657 (1971), and Geiger et al., Bio-chem. Biophys. Res. Com. 55, 60-66 (1973)).
In the context of genetic engineering, it is now possible to prepare insulin from microorganisms modified by genetic engineering. If E. coli is used as the micro-organism, the product is frequently expressed as fusion protein, that is to say the product is coupled with a protein endogenous to the bacteria, for example with ~-galactosidase. This fusion protein precipitates out in the cell and is in this way protected from proteolytic degradation. After breakdown of the cell, the fusion protein content i8 split off chemically or enzymatically and the 6 cysteines of the insulin precursor are con-verted into their S-sulfonates (-S-S03- ) by means of oxidative sulfitolysis. In a subsequent ("renaturing or recombination~) step, natural preproinsulin must be produced from this so-called preproinsulin S-sulfonate by formation of the 3 correct disulfide bridges - that is to ~ L~ 6 say -S-S- bridges from A6 to All, from A7 to B7 and from A20 to Bl9 in the corresponding insulin peptide sequen-ces.
According to the process described in EP-B-0,037,255, this step is carried out, for example, by reaction of the starting S-sulfonate with a mercaptan in an amount which results in 1 to 5 SH radicals per SS03- radical in an aqueous medium at a pH of 7 to 11.5 and an S-sulfonate concentration of up to 10 mg per ml of aqueous medium, preferably in the absence of an oxidizing agent.
Yields of in some cases more than 80% are said to be obtained here.
In addition to the (desired) renaturing products with correct disulfide bridges, more or less substantial amounts of ~undesired) "incorrect" recombinants, that is to say insulin products with disulfide bridges which are only partly correct or not correct at all and also with intermolecular disulfide bridges, are always also formed - depending on the reaction conditions and in particular the concentration circumstances - in this and practically all other known renaturing or recombination processes in which insulin precursors with opened disulfide bridges are converted into products with the correspondingly closed disulfide bridges.
When the insulin precursor products obtained by the known renaturing processes are worked up to insulin without the "incorrect" recombinants being removed - this working up being effected by known techniques (chemically or enzymatically) - no (natural) insulin is formed from the "incorrect" recombinants.
It is therefore advantageous or necessary to remove the ~incorrect" recombinants from the renaturing products with the correct disulfide bridges before working up the corresponding renaturing products to give insulin. This ~0(~ 4fi can be effected, for example, by known chromatographic processes. In another particularly advantageous process, the removal is effected by adjusting the reaction mixture to pH 4 to 6 - preferably in the presence of a small amount of a physiologically acceptable surface-active substance - the ~correct recombinants remaining virtu-ally completely in solution and the "incorrect" recom-binants being precipitated (c.f. DE-A-35 01 641).
The "incorreck" recombinants removed are then advantage-ously converted back by sulfitolysis into the correspond-ing S-sulfonate, which is subjected to renewed folding, it often being necessary to remove by-products formed during the sulfitolysis by chromatography before the renaturing. The ~incorrect recombinants can in this way largely be converted back into "usable" product.
Sulfitolysis of the "incorrect" recombinants with subse-quent chromatography and renewed folding of course means a not inconsiderable expenditure.
In the efforts to avoid or at least reduce this expendi-ture, it has now been found that this is possible byreacting the "incorrect" recombinants of insulin precur-sors with excess mercaptan in an aqueous medium in the presence of an organic redox system or at least one organic compound which forms such an organic redox system under the reaction conditions.
The "incorrect" recombinants can in this way be converted in high yields directly into the "correct" renaturing products or "correct" recombinants without sulfitolysis, which represents a considerable advantage compared with the prior art.
There is also no prior art at all which would have made obvious in any manner bypassing of sulfitolysis with the subsequent folding stage merely by reaction with excess mercaptan and the addition of an organic redox system.
~OC~
Possible incorrect recombinants of insulin precursors for the process accordin~ to the invention are preferably the products which are formed as by-products on recom-bination of insulin precursors with opened -S-S- bridges of the following formula I:
(A-1) Gly-NH -- X
I
(A-6) Cys-S-R3 S-R3 I l (A-20) (A-21) (A-7) Cys-------Cys--------- Cys - Z - R2 I (A~ (I) (3-1) 1 l R1-HN-Phe---Cys------------------~ Cys--------------Y
~B-7) (B-19) (B-30) in which R~ = H or an amino acid or peptide radical which can be split off chemically or enzymatically, R2 = OH or an amino acid or peptide radical, preferably OH, R3 - H or a Cy~-5 protective group, preferably the -SO3- or the tert.-butyl group, X = a radical which ~oins the insulin A and B
chains, preferably an amino acid or peptide radical, Y = the radical of a genetically encodable amino acid, preferably Thr, Ala or Ser, in particular Thr, Z = the radical of a genetically encodable amino acid, preferably Asn, Gln, Asp, Glu, Gly, Ser, Thr, Ala or Met, in particular Asn, and ~l-A2Q and Bl-B29 = peptide sequences of insulin which are non-mutated or mutated by re-placement of one or more amino acids, - s -preferably the non-mutated peptide sequen-ces of human, porcine or bovine insulin, in particular of human or porcine insulin.
If R, = H in formula I, the products are products which are derived from proinsulin; if Rl = an amino acid or peptide radical which can be split off chemically or enzymatically, the products are products which are derived from preproinsulin.
Amino acid radicals which can be split off chemically are those which are split off, for example, by means of BrCN
or N-bromosuccinimide; these are, for example, methionine (Met) or tryptophan (Trp).
Amino acid radicals which can be split off enzymatically are those which can be split off, for example, by means of trypsin (such as, for example, Arq or Lys).
Peptide radicals which can be split off chemically or enzymatically are peptide radica's having at least 2 amino acids.
All the amino acids possible for Rl are preferably those of the group of naturally occurring amino acids, that is to ~ay mainly Gly, Ala, Ser, Thr, Val, Leu, Ile, Asn, Gln, Cys, Met, Tyr, Phe, Pro, Hyp, Arg, Lys, Hyl, Orn, Cit and His.
R2 is OH or - sLmilarly to Rl - likewise an amino acid or peptide radical, the meaning of OH being preferred. The amino acids (including those which form the peptide radical - consisting of at ~east 2 amino acid radicals) preferably originate - as for Rl - from the group of naturally occurring amino acids.
R3 is hydrogen or a cysteine-sulfur protective group, the -S03- or the tert.-butyl group being preferred cysteine-sulfur protective groups.
;~0~ 6 X is a radical which joins the insulin A and B chains, preferably an amino acid or peptide radical.
If X is an amino acid radical, the radical of Arg or Lys is preferred; if X is a peptide radical, the radical of a naturally occurring C-peptide - in particular the human, porcine or bovine insulin C-peptide - is preferred.
Genetically encodable amino acids - for Y - are (in each case in the L form): Gly, Ala, Ser, Thr, Val, Leu, Ile, Asp, Asn, Glu, Gln, Cys, Met, Arg, Lys, His, Tyr, Phe, Trp and Pro.
Preferred genetically encodable amino acids are Thr, Ala and Ser, in particular Thr.
Z can - like Y - also denote the radical of a genetically encodable amino acid, but in this case Asn, Gln, Asp, Glu, Gly, Ser, Thr, Ala and Met, in particular Asn, are preferred.
Al - A20 and Bl - B29 can in principle be the peptide sequences which are non-mutated or mutated by replacement of one or more amino acids and originate from all pO8-sible insulins; the mutants can be produced by known processes of genetic engineering (site directed mutagene-sis~. However, the non-mutated peptide sequences of human, porcine or bovine insulin, in particular of human or porcine insulin ~the Al - A20 and B1 - B29 sequences of human and porcine insulin are identical) are preferred.
The "incorrect" recombinants formed as by-products during recombination of insulin precursors with opened -S-S-bridges of the formula I are removed from the "correct"
recombinants in a known manner - preferably by precipita-tion at pH values of ~ to 6 in accordance with the process of the abovementioned DE-A-35 01 641 - and are X0(~46 then dissolved directly, or after prior freeze-drying, in water or an aqueous solution for use for the process according to the invention.
The concentration of the "incorrect" recombinants in the aqueous starting solution can vary within a wide range;
preferred concentrations are about 0.1 to about 100 mg, in particular about 0.1 to about 10 mg/ml, the mg values relating to the "incorrect~ recombinants as dried solid.
Suitable mercaptans for the reaction according to the invention are in principle all the possible organic compounds with SH groups; mercaptoethanol, thioglycolic acid, dithioerythritol, qlutathione and cysteine, in particular mercaptoethanol and cysteine, are preferred.
The mercaptans can be employed individually or as a mixture.
The amount of mercaptan to be employed can vary within wide limits; an excess of mercaptan corresponding to a ratio of mercaptan-SH groups~cysteine-S units (in the ~incorrect~' recombinants) of at least about 5 ifi pre-ferred. This ratio has an upper limit imposed practicallyonly by economic considerations. An upper limit of about 100 i8 advantageous.
Because of the wide range of variation of the excess mercaptan, the number of cysteine-S units in the "in-correct" recombinant employed does not have to be deter-mined completely accurately.
Preferred possible organic redox systems are pairs of compounds, one component of which is an organic compound having the structural element of the formula II
OH OH O
1~ (II) O OH OH
-- C -- C _ C --ZOG~ 6 or an aromatic o- or p-dihydroxy compound and the other component of which is an organic compound having the structural element of the formula II in oxidized form =
structural element of the formula II' O O O
5- C - C - C - (II') or is an o- or p-quinone.
The free valencies of the structural element of the formula II and II' can be satisfied by hydrogen or organic groups, such as, for example, C1-C4-alkyl groups.
However, ~he structural element can also be part of a ring having preferably 4, 5 or 6 carbon ring atoms and if appropriate also one or two hetero atoms, such as, for example, O, it being possible for the ring in turn to be substituted by groups which are inert under the reaction conditions, such as, for example, alkyl or hydroxyalkyl groups.
Examples of compounds having the structural element of the formula II are:
reductone OH OH O
H - C = C - C - H
reductic acid pH OH
HC - CH
\C' o methylreductic acid OH OH
H~ -- CH
C O
X~ 6 g ascorbic acid OH OH
(vitamin C) OH HC CH
O
The formulae here are in each case written only in one of the tautomeric forms.
All the compounds are reducing. In the oxidized form, the structural element of the formula II becomes that of the formula II'.
Possible aromatic o- and p-dihydroxy compounds are in principle all the possible aromatic compounds having two OH groups in the o- or p-position, it merely being necessary that the o- or p-quinone formation from the o-and p-dihydroxy compounds cannot be prevented by any particular substituents or the like. Examples of aromatic o- and p-dihydroxy compounds are 1,2-dihydroxybenzene = pyrocatechol, 1,4-dihdyroxybenzene = hydroquinone, methyl-hydroquinone, naphtho-1,4-hydroxyguinone and anthra-hydroquinone; on oxidation, the corresponding quinones are formed therefrom.
In the reaction according to the invention the particular organic redox systems consisting, for example, of the pairs of compounds of ascorbic acid + dehydroascorbic acid, pyrocatechol + o-quinone, hydroquinone + p-quinone, naphthohydroquinone + naphthoquinone and the like, can thus be employed in virtually any desired ratio (prefer-ably in an approximately equimolar ratio). However, it is also possible for only the particular individual com-ponen~s of these pairs of compounds - that is to say, for example, only ascorbic acid or only dehydroascorbic acid or only hydroquinone and the like - to be added, because the other particular component belonying to the redox pair of compounds ~dehydroascorbic acid or ascorbic acid or p-quinone and the like) forms in the reaction medium.
x~ >~
Preferred organic redox systems are the combinations consisting of the pairs of compounds ascorbic acid + dehydroascorbic acid, pyrocatechol + o-quinone and hydroquinone + p-quinone and preferred individual compounds which form such a redox system under the reaction conditions are the individual components of these pairs of compounds.
Ascorbic acid and/or dehydroascorbic acid are especially preferred.
The amount employed of the compound(s) which form(s) the organic redox system can vary within wide limits. The number of mol of compound(s) which fo~m(s) the organic redox system can be chosen as being between about 1/10,000 and 10,000, preferably between about 1/10 and 10, based on one gram equivalent of mercaptan (= mole-cular weight of the mercaptan employed in g/number of SH
groups in the mercaptan molecule)~
It is advantageous also to add urea to the reaction solution, concentrations corresponding to about 0.1 to 1 M ~M = molar), in particular 0.1 - 0.5 M, being pre-ferred.
The reaction according to the invention is advantageously carried out in the alkaline pH range, preferably between about 7 and 12, in particular between about 9.5 and 11.
To maintain the desired pH, it is advantageous to add a buffer substance, the nature and ionic strength of the buffer having a certain influence on the folding yield.
It is advantageous to keep the ionic strength low, a 3~ range of about 1 mM (mM = millimolar) to 1 M (M = molar), in particular one from about 5 mM to 50 mM, being pre-ferred. Buffer substances which can be used are, for example, borate buffer, carbonate buffer or glycine buffer, the latter being preferred.
XO~ ~4fi A general range of reaction temperature which may be stated is one between about 0 and 45C; a range from about 4 to 8DC is preferred.
Covering the renaturing solution with a layer of certain gases, such as, for example, oxygen, nitrogen or helium, has no noticeable influence on the renaturing yield.
The reaction time is in general between about ~ and 24 hours, preferably between about 6 and 16 hours.
The renaturing product of the reaction according to the invention is - if the "incorrect" starting recombinant originates from recombination of an insulin precursor with opened S-S bridges of the abovementioned formula I -an insulin precursor having correct disulfide bonds (~correct" recombinant) of the formula III
(A-1) Gly-NH X
I
(A-6) Cys-S-S
I ¦ (A-20) (A-21) (A-7) Cys---Cys--------- Cys - Z - R2 ¦ (A-11) l (III) S S
(B-1) l l Rl-HN-Phe---Cys--------------- Cy~------------Y
(~-7) tB-19) (B-30) in which Rl, R2, X, Y and Z have the same meaning as in formula I.
When the reaction has ended (which can be ascertained, - for example, by high performance liquid chromatography), the mixture is worked up in a known manner.
The "correctly" folded product, preferably of the formula III, can then be converted into the corresponding insulin enzymatically or chemically by known techniques.
~o~
The following example is intended to illustrate the invention in more detail. Before the (invention) example, the preparation of the starting substance is also des-cribed by way of example.
A) Preparation of the starting substance 1. Folding of "miniproinsulin"
~Miniproinsulin-S-SO3~, that is to say an insulin precursor in which the A and B chains of the insulin are linked via an arginine and the B chain is leng-thened N-terminally, is employed. The freeze-dried material (60% pure) is dissolved at a solids con-centration of 0.5 g/l in 50 mM glycine buffer, pH
10.7, which corresponds to a precursor concentration of 0.3 g/l. 630 ml of 1 M mercaptoethanol and 630 ml of 1 M ascorbic acid are added to the batch (100 1), and the mixture is then stirred slowly in a cold chamber at 8C for 16 hours. The folding yield, determined by high performance liquid chromatography against a standard, is 0.228 g/l (76% of theory).
2. Precipitation of aggregates ("incorrect" recombinants) 1 g of polyethylene-polypropylene glycol is added to the folding batch and the total batch is divided into 5 batches of 20 ml, with which a pH precipitation series between pH 5.0 and pH 7.0 is set up in 0.5 pH
value steps. After establishing the pH values, the mixtures are left to stand at room temperature for 15 minutes and the precipitates are then centrifuged off.
The supernatants are quantified by means of high performance liquid chromatography in order to deter-mine the precipitation losses, and the precipitates are combined ~nd freeze-dried (weight: 15 g of solid, content 40% pure).
Loss of correctly folded product as a function of the pH:
20~~
pH 5.0 0~
pH 5.5 6%
pH 6.0 10 pH 6.5 9 pH 7.0 4~
This series shows that the optimum precipitation pH is 5Ø
B) Example according to the invention: Renaturing 15 g of freeze-dried precipitate are taken up in 5 1 of ~ M urea. 13.2 ml of mercaptoethanol (14.35 M) are added (final concentration about 35 mM) and the mixture is left at room temperature for 10 minutes. The 5 1 of solution are introduced into 25 1 of 50 mM glycine buffer, 188 ml of 1 M ascorbic acid are added and the pH is brought to 10.7. The batch iæ then stirred gently at 8C for 5 hours. The folding yield is 0.146 g/l (73% of theory).
The course of the reaction i~ monitored by means of high performance liquid chromatography.
The total yield of the "miniproinsulin" folding (c.f. A1) can thus be increased from 76~ of theory to about 85% of theory.
value steps. After establishing the pH values, the mixtures are left to stand at room temperature for 15 minutes and the precipitates are then centrifuged off.
The supernatants are quantified by means of high performance liquid chromatography in order to deter-mine the precipitation losses, and the precipitates are combined ~nd freeze-dried (weight: 15 g of solid, content 40% pure).
Loss of correctly folded product as a function of the pH:
20~~
pH 5.0 0~
pH 5.5 6%
pH 6.0 10 pH 6.5 9 pH 7.0 4~
This series shows that the optimum precipitation pH is 5Ø
B) Example according to the invention: Renaturing 15 g of freeze-dried precipitate are taken up in 5 1 of ~ M urea. 13.2 ml of mercaptoethanol (14.35 M) are added (final concentration about 35 mM) and the mixture is left at room temperature for 10 minutes. The 5 1 of solution are introduced into 25 1 of 50 mM glycine buffer, 188 ml of 1 M ascorbic acid are added and the pH is brought to 10.7. The batch iæ then stirred gently at 8C for 5 hours. The folding yield is 0.146 g/l (73% of theory).
The course of the reaction i~ monitored by means of high performance liquid chromatography.
The total yield of the "miniproinsulin" folding (c.f. A1) can thus be increased from 76~ of theory to about 85% of theory.
Claims (13)
1. A process for renaturing "incorrect" recombinants of an insulin precursor, which comprises reacting the "incorrect" recombinant with excess mercaptan in an aqueous medium in the presence of an organic redox system or at least one organic compound which forms such an organic redox system under the reaction conditions.
2. The process as claimed in claim 1, wherein the "incorrect" recombinants of an insulin precursor used are the products which are formed as by-pro-ducts on recombination of an insulin precursor with opened -S-S- bridges of the formula I
(I) in which R1 = H or an amino acid or peptide radical which can be split off chemically or enzymatically, R2 = OH or an amino acid or peptide radical, preferably OH, R3 = H or a Cys-S protective group, preferably the -SO3- or the tert.-butyl group, X = a radical which joins the insulin A and B
chains, preferably an amino acid or peptide radical, Y = the radical of a genetically encodable amino acid, preferably Thr, Ala or Ser, in particular Thr, Z = the radical of a genetically encodable amino acid, preferably Asn, Gln, Asp, Glu, Gly, Ser, Thr, Ala or Met, in particular Asn, and A1-A20 and B1-B29 = peptide sequences of insulin which are non-mutated or mutated by re-placement of one or more amino acids, preferably the non-mutated peptide sequen-ces of human, porcine or bovine insulin, in particular of human or porcine insulin.
(I) in which R1 = H or an amino acid or peptide radical which can be split off chemically or enzymatically, R2 = OH or an amino acid or peptide radical, preferably OH, R3 = H or a Cys-S protective group, preferably the -SO3- or the tert.-butyl group, X = a radical which joins the insulin A and B
chains, preferably an amino acid or peptide radical, Y = the radical of a genetically encodable amino acid, preferably Thr, Ala or Ser, in particular Thr, Z = the radical of a genetically encodable amino acid, preferably Asn, Gln, Asp, Glu, Gly, Ser, Thr, Ala or Met, in particular Asn, and A1-A20 and B1-B29 = peptide sequences of insulin which are non-mutated or mutated by re-placement of one or more amino acids, preferably the non-mutated peptide sequen-ces of human, porcine or bovine insulin, in particular of human or porcine insulin.
3. The process as claimed in either of claims 1 and 2, wherein the reaction is carried out at concentra-tions of the "incorrect" recombinants of about 0.1 to about 100mg, preferably about 0.1 to about 10 mg/ml.
4. The process as claimed in any one of claims 1 to 3, wherein mercaptoethanol and/or cysteine is used as the mercaptan.
5. The process as claimed in any one of claims 1 to 4, wherein the reaction is carried out with an excess of mercaptan corresponding to a ratio of mercaptan-SH groups/cysteine-S units (in the "incorrect"
recombinants) of at least about 5, preferably about 5 to 100.
recombinants) of at least about 5, preferably about 5 to 100.
6. The process as claimed in any one of claims 1 to 5, wherein the organic redox system used is a pair of compound, one component of which is an organic compound having the structural element of the formula II
(II) or an aromatic o- or p-dihydroxy compound and the other component of which is an organic compound having the structural element of the formula II in oxidized form = structural element of the formula II' (II) or is an o- or p-quinone.
(II) or an aromatic o- or p-dihydroxy compound and the other component of which is an organic compound having the structural element of the formula II in oxidized form = structural element of the formula II' (II) or is an o- or p-quinone.
7. The process as claimed in any one of claims 1 to 5, wherein the organic compounds employed which form an organic redox system under the reaction conditions are one or more of the individual components men-tioned in claim 6.
8. The process as claimed in any one of claims 1 to 7, wherein the organic redox system employed is the pair of compounds ascorbic acid + dehydroascorbic acid, pyrocatechol + o-quinone or hydroquinone + p-quinone and the organic compound employed which can form such a redox system under the reaction conditions is in each case only one component of this pair of compounds.
9. The process as claimed in any one of claims 1 to 7, wherein the reaction is carried out in the presence of ascorbic acid and/or dehydroascorbic acid.
10. The process as claimed in any one of claims 1 to 9, wherein the mercaptan and the compound(s) which form(s) the organic redox system are employed in a ratio of 1 gram equivalent of mercaptan to 1/10,000 to 10,000, preferably 1/10 to 10 mol of the com-pound(s) which form(s) the organic redox system.
11. The process as claimed in any one of claims 1 to 10, wherein the aqueous reaction medium contains dis-solved urea, preferably in an approximately 0.1 - 1, in particular approximately 0.1-0.5 molar concentra-tion.
12. The process as claimed in any one of claims 1 to 11, wherein the reaction is carried out at a pH of between about 7 and 12, preferably between about 9.5 and 11.
13. The process as claimed in claim 1, and substantially as described herein.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE3901718A DE3901718A1 (en) | 1989-01-21 | 1989-01-21 | METHOD FOR RENATURING INCORRECT RECOMBINANT OF INSULIN PREFERRED |
DEP3901718.4 | 1989-01-21 |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2008246A1 true CA2008246A1 (en) | 1990-07-21 |
Family
ID=6372511
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002008246A Abandoned CA2008246A1 (en) | 1989-01-21 | 1990-01-22 | Process for renaturing incorrect recombinants of insulin precursor |
Country Status (12)
Country | Link |
---|---|
EP (1) | EP0379162A3 (en) |
JP (1) | JPH02233698A (en) |
AU (1) | AU628473B2 (en) |
CA (1) | CA2008246A1 (en) |
DE (1) | DE3901718A1 (en) |
FI (1) | FI900295A0 (en) |
HU (1) | HU207526B (en) |
IL (1) | IL93114A0 (en) |
NO (1) | NO900277L (en) |
NZ (1) | NZ232177A (en) |
PT (1) | PT92906A (en) |
ZA (1) | ZA90395B (en) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE3901719A1 (en) * | 1989-01-21 | 1990-07-26 | Hoechst Ag | METHOD FOR PRODUCING AN INSULIN PREVENTE |
AU664021B2 (en) * | 1990-09-05 | 1995-11-02 | Natinco Nv | Solubilization of proteins in active forms |
US6001604A (en) * | 1993-12-29 | 1999-12-14 | Bio-Technology General Corp. | Refolding of proinsulins without addition of reducing agents |
DE4405179A1 (en) * | 1994-02-18 | 1995-08-24 | Hoechst Ag | Method of obtaining insulin with correctly connected cystine bridges |
PT871474E (en) † | 1994-12-29 | 2007-02-28 | Ferring Int Ct Sa | Generation of human insulin |
PL212317B1 (en) | 2003-01-31 | 2012-09-28 | Organon Nv | Method for protein isolation in anoxic conditions |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA1154435A (en) * | 1980-03-27 | 1983-09-27 | Bruce H. Frank | Process for producing an insulin precursor |
DE3501641A1 (en) * | 1985-01-19 | 1986-07-24 | Hoechst Ag, 6230 Frankfurt | METHOD FOR OBTAINING INSULIN PRECURSORS FROM REACTION MIXTURES WHICH ARE INCLUDED IN THE FOLDING OF INSULIN PRECURSORS FROM THE CORRESPONDING S-SULPHONATES |
DE3901719A1 (en) * | 1989-01-21 | 1990-07-26 | Hoechst Ag | METHOD FOR PRODUCING AN INSULIN PREVENTE |
-
1989
- 1989-01-21 DE DE3901718A patent/DE3901718A1/en not_active Withdrawn
-
1990
- 1990-01-17 EP EP19900100890 patent/EP0379162A3/en not_active Withdrawn
- 1990-01-18 FI FI900295A patent/FI900295A0/en not_active Application Discontinuation
- 1990-01-19 NZ NZ232177A patent/NZ232177A/en unknown
- 1990-01-19 NO NO90900277A patent/NO900277L/en unknown
- 1990-01-19 PT PT92906A patent/PT92906A/en unknown
- 1990-01-19 JP JP2008567A patent/JPH02233698A/en active Pending
- 1990-01-19 AU AU48620/90A patent/AU628473B2/en not_active Ceased
- 1990-01-19 IL IL93114A patent/IL93114A0/en unknown
- 1990-01-19 HU HU90196A patent/HU207526B/en not_active IP Right Cessation
- 1990-01-19 ZA ZA90395A patent/ZA90395B/en unknown
- 1990-01-22 CA CA002008246A patent/CA2008246A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
FI900295A0 (en) | 1990-01-18 |
ZA90395B (en) | 1990-09-26 |
NZ232177A (en) | 1991-11-26 |
EP0379162A3 (en) | 1991-10-16 |
AU628473B2 (en) | 1992-09-17 |
HUT54179A (en) | 1991-01-28 |
AU4862090A (en) | 1990-07-26 |
DE3901718A1 (en) | 1990-07-26 |
JPH02233698A (en) | 1990-09-17 |
NO900277D0 (en) | 1990-01-19 |
NO900277L (en) | 1990-07-23 |
IL93114A0 (en) | 1990-11-05 |
HU900196D0 (en) | 1990-03-28 |
EP0379162A2 (en) | 1990-07-25 |
PT92906A (en) | 1990-07-31 |
HU207526B (en) | 1993-04-28 |
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