WO2024112250A1 - Method of preparing vasopressin - Google Patents
Method of preparing vasopressin Download PDFInfo
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- WO2024112250A1 WO2024112250A1 PCT/SE2023/051176 SE2023051176W WO2024112250A1 WO 2024112250 A1 WO2024112250 A1 WO 2024112250A1 SE 2023051176 W SE2023051176 W SE 2023051176W WO 2024112250 A1 WO2024112250 A1 WO 2024112250A1
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- WIPO (PCT)
- Prior art keywords
- vasopressin
- ethanol
- acetic acid
- solution
- cys
- Prior art date
Links
- 108010004977 Vasopressins Proteins 0.000 title claims abstract description 96
- 102000002852 Vasopressins Human genes 0.000 title claims abstract description 95
- KBZOIRJILGZLEJ-LGYYRGKSSA-N argipressin Chemical compound C([C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@@H](C(N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N1)=O)N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(N)=O)C1=CC=CC=C1 KBZOIRJILGZLEJ-LGYYRGKSSA-N 0.000 title claims abstract description 80
- 229960003726 vasopressin Drugs 0.000 title claims abstract description 76
- GXBMIBRIOWHPDT-UHFFFAOYSA-N Vasopressin Natural products N1C(=O)C(CC=2C=C(O)C=CC=2)NC(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CCCN=C(N)N)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C1CC1=CC=CC=C1 GXBMIBRIOWHPDT-UHFFFAOYSA-N 0.000 title claims abstract description 73
- 238000000034 method Methods 0.000 title claims abstract description 48
- 239000011347 resin Substances 0.000 claims abstract description 51
- 229920005989 resin Polymers 0.000 claims abstract description 51
- 238000000746 purification Methods 0.000 claims abstract description 27
- JLTCWSBVQSZVLT-UHFFFAOYSA-N n-[6-amino-1-[(2-amino-2-oxoethyl)amino]-1-oxohexan-2-yl]-1-[19-amino-7-(2-amino-2-oxoethyl)-10-(3-amino-3-oxopropyl)-13-benzyl-16-[(4-hydroxyphenyl)methyl]-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentazacycloicosane-4-carbonyl]pyrrolidine-2-carboxa Chemical compound NCCCCC(C(=O)NCC(N)=O)NC(=O)C1CCCN1C(=O)C1NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C(CC=2C=CC=CC=2)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(N)CSSC1.N1C(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CCCN=C(N)N)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C1CC1=CC=CC=C1 JLTCWSBVQSZVLT-UHFFFAOYSA-N 0.000 claims abstract description 21
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 154
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 88
- 239000000243 solution Substances 0.000 claims description 45
- 238000004366 reverse phase liquid chromatography Methods 0.000 claims description 43
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 21
- 239000005695 Ammonium acetate Substances 0.000 claims description 21
- 229940043376 ammonium acetate Drugs 0.000 claims description 21
- 235000019257 ammonium acetate Nutrition 0.000 claims description 21
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 16
- 239000007864 aqueous solution Substances 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 12
- 239000002253 acid Substances 0.000 claims description 10
- 238000010828 elution Methods 0.000 claims description 10
- 239000000539 dimer Substances 0.000 claims description 8
- 239000000377 silicon dioxide Substances 0.000 claims description 8
- 150000001875 compounds Chemical class 0.000 claims description 7
- 239000002243 precursor Substances 0.000 claims description 7
- 150000003573 thiols Chemical group 0.000 claims description 7
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 5
- 229910052740 iodine Inorganic materials 0.000 claims description 5
- 239000011630 iodine Substances 0.000 claims description 5
- 230000002378 acidificating effect Effects 0.000 claims description 4
- 150000007529 inorganic bases Chemical class 0.000 claims description 4
- 239000007800 oxidant agent Substances 0.000 claims description 4
- 230000003472 neutralizing effect Effects 0.000 claims description 3
- 159000000021 acetate salts Chemical class 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 239000008194 pharmaceutical composition Substances 0.000 claims description 2
- 239000007787 solid Substances 0.000 claims description 2
- 238000011210 chromatographic step Methods 0.000 claims 6
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 claims 3
- GNFTZDOKVXKIBK-UHFFFAOYSA-N 3-(2-methoxyethoxy)benzohydrazide Chemical compound COCCOC1=CC=CC(C(=O)NN)=C1 GNFTZDOKVXKIBK-UHFFFAOYSA-N 0.000 claims 1
- 150000003839 salts Chemical class 0.000 claims 1
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 32
- 238000010511 deprotection reaction Methods 0.000 abstract description 18
- 239000007790 solid phase Substances 0.000 abstract description 10
- 238000010647 peptide synthesis reaction Methods 0.000 abstract description 9
- 238000003776 cleavage reaction Methods 0.000 abstract description 8
- 230000003647 oxidation Effects 0.000 abstract description 8
- 238000007254 oxidation reaction Methods 0.000 abstract description 8
- 230000007017 scission Effects 0.000 abstract description 8
- 125000004122 cyclic group Chemical group 0.000 abstract description 5
- 238000002955 isolation Methods 0.000 abstract description 4
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 30
- 239000012071 phase Substances 0.000 description 14
- -1 acetamidomethyl Chemical group 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 239000008186 active pharmaceutical agent Substances 0.000 description 11
- 150000001413 amino acids Chemical class 0.000 description 11
- 239000003153 chemical reaction reagent Substances 0.000 description 11
- 238000005859 coupling reaction Methods 0.000 description 11
- 229940088679 drug related substance Drugs 0.000 description 11
- 125000006239 protecting group Chemical group 0.000 description 11
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 10
- 239000002904 solvent Substances 0.000 description 10
- 235000001014 amino acid Nutrition 0.000 description 9
- 238000010168 coupling process Methods 0.000 description 9
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 8
- 230000008878 coupling Effects 0.000 description 8
- 239000012535 impurity Substances 0.000 description 8
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 8
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 7
- 230000021736 acetylation Effects 0.000 description 7
- 238000006640 acetylation reaction Methods 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 6
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 6
- 230000005526 G1 to G0 transition Effects 0.000 description 5
- 238000002835 absorbance Methods 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 4
- 229960004592 isopropanol Drugs 0.000 description 4
- 238000004886 process control Methods 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 239000008213 purified water Substances 0.000 description 4
- VXGGBPQPMISJCA-STQMWFEESA-N (2s)-2-[[(2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)propanoyl]amino]propanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O)C3=CC=CC=C3C2=C1 VXGGBPQPMISJCA-STQMWFEESA-N 0.000 description 3
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 description 3
- 150000003862 amino acid derivatives Chemical class 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000012792 lyophilization process Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000006386 neutralization reaction Methods 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 239000002516 radical scavenger Substances 0.000 description 3
- UGNWTBMOAKPKBL-UHFFFAOYSA-N tetrachloro-1,4-benzoquinone Chemical compound ClC1=C(Cl)C(=O)C(Cl)=C(Cl)C1=O UGNWTBMOAKPKBL-UHFFFAOYSA-N 0.000 description 3
- BJFIDCADFRDPIO-DZCXQCEKSA-N (2S)-N-[(2S)-6-amino-1-[(2-amino-2-oxoethyl)amino]-1-oxohexan-2-yl]-1-[[(4R,7S,10S,13S,16S,19R)-19-amino-7-(2-amino-2-oxoethyl)-10-(3-amino-3-oxopropyl)-16-[(4-hydroxyphenyl)methyl]-6,9,12,15,18-pentaoxo-13-(phenylmethyl)-1,2-dithia-5,8,11,14,17-pentazacycloeicos-4-yl]-oxomethyl]-2-pyrrolidinecarboxamide Chemical compound NCCCC[C@@H](C(=O)NCC(N)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC=2C=CC(O)=CC=2)NC(=O)[C@@H](N)CSSC1 BJFIDCADFRDPIO-DZCXQCEKSA-N 0.000 description 2
- 125000004217 4-methoxybenzyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1OC([H])([H])[H])C([H])([H])* 0.000 description 2
- 101800001144 Arg-vasopressin Proteins 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 108010048179 Lypressin Proteins 0.000 description 2
- 101100252610 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) rpc-82 gene Proteins 0.000 description 2
- 102100026383 Vasopressin-neurophysin 2-copeptin Human genes 0.000 description 2
- 239000003929 acidic solution Substances 0.000 description 2
- 150000001408 amides Chemical class 0.000 description 2
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 2
- 239000012045 crude solution Substances 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 239000012351 deprotecting agent Substances 0.000 description 2
- 125000002228 disulfide group Chemical group 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 229960003837 lypressin Drugs 0.000 description 2
- 230000001473 noxious effect Effects 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- HLCTYBOTPCIHTG-QMMMGPOBSA-N (2r)-3-(acetamidomethylsulfanyl)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound CC(=O)NCSC[C@@H](C(O)=O)NC(=O)OC(C)(C)C HLCTYBOTPCIHTG-QMMMGPOBSA-N 0.000 description 1
- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-N 0.000 description 1
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- DFPYXQYWILNVAU-UHFFFAOYSA-N 1-hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1.C1=CC=C2N(O)N=NC2=C1 DFPYXQYWILNVAU-UHFFFAOYSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- OXDZADMCOWPSOC-UHFFFAOYSA-N Argiprestocin Chemical class N1C(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CCCN=C(N)N)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C(C(C)CC)NC(=O)C1CC1=CC=C(O)C=C1 OXDZADMCOWPSOC-UHFFFAOYSA-N 0.000 description 1
- XUJNEKJLAYXESH-UWTATZPHSA-N D-Cysteine Chemical compound SC[C@@H](N)C(O)=O XUJNEKJLAYXESH-UWTATZPHSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical compound [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000006167 equilibration buffer Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000008571 general function Effects 0.000 description 1
- 231100001261 hazardous Toxicity 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000010829 isocratic elution Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- COTNUBDHGSIOTA-UHFFFAOYSA-N meoh methanol Chemical compound OC.OC COTNUBDHGSIOTA-UHFFFAOYSA-N 0.000 description 1
- 150000002730 mercury Chemical class 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- XNOPRXBHLZRZKH-MQYCRUOZSA-N oxytocin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@H](N)C(=O)N1)C(=O)N1C(CCC1)C(=O)N[C@@H](CC(C)C)C(=O)NCC(N)=O)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 XNOPRXBHLZRZKH-MQYCRUOZSA-N 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 150000003140 primary amides Chemical class 0.000 description 1
- 230000009103 reabsorption Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000012508 resin bead Substances 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 229910000077 silane Inorganic materials 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 1
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 238000007039 two-step reaction Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000010626 work up procedure Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/16—Oxytocins; Vasopressins; Related peptides
Definitions
- the present invention relates to a new method of preparing vasopressin.
- the vasopressins are hormone peptides consisting of nine amino acids (nonapeptides) and has a general function of regulating the tonicity of body fluids and control water reabsorption.
- the amino acid sequence of human vasopressin (antidiuretic hormone (ADH)) or arginine vasopressin (argipressin or AVP) is Cys- Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2, with the cysteine residues forming a disulfide bond and the C-terminus of the sequence converted to a primary amide.
- Lysine vasopressin has a lysine in place of the arginine as the eighth amino acid, and is found in pigs and some related animals.
- SPPS solid phase peptide synthesis
- CN106632615B CN106699851 B; CN106518978B; CN109942686A; CN1 10016071 B; CN110003313B; CN110016070A; CN109929011 B; CN1 10078795A; W02020224580; IN201841030341 ; W02020212477; CN106866744B; CN109836470A; and IN201841035862A.
- WO 2006/041945 discloses a method of producing vasopressin with Fmoc based SPPS using a two-step reaction to cleave and deprotect the resin bound vasopressin precursor nonapeptide and a following complicated and consuming work-up process with ether precipitation, filtration, drying, dissolution before purification of linear vasopressin precursors.
- orthogonal protecting group refers to a protecting group of an amino acid side chain that is chemically resistant under one set of selected conditions but is liable under another set of conditions.
- Orthogonal protecting groups for thiol containing residues include, but are not limited to, at least one of acetamido methyl (Acm), benzyl (Bzl), 4-methoxybenzyl (Mob), tertbutyl (Tbu or t-Bu), trimethylacetamidomethyl (Tacm), phenylacetamidomethyl (Phacm), or tert-butylmercapto (StBu).
- the orthogonal protecting group is a non-acid labile group such as acetamido methyl.
- the peptide chain is not completely deprotected after deprotection or cleavage but carries protecting groups attached to the thiol containing residues through a first purification step.
- scavengers relates to organic compounds added to the cleavage and deprotection step of resin bound protected peptide to quench formed reactive ions, especially cations during this process step.
- Such compounds include silane scavengers such as triisopropylsilane and thiol scavengers such as dithiothreitol (DTT), anisole, thioanisole or 1 ,2-ethanedithiol.
- DTT dithiothreitol
- Free from scavengers in the context of the present context means free from this type of organic scavengers that may form impurities difficult to remove with following purification steps.
- Boc(1-9)resin vasopressin is the protected linear peptide, not cleaved from the resin: Boc-Cys(Acm)-Tyr(tBu)-Phe-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro- Arg(Pbf)-Gly-Rink-AM-resin.
- H(1-9)NH2 vasopressin neutralized solution is the neutralized peptide, cleaved from the resin and partly deprotected in solution, not purified: H- Cys(Acm)-Tyr-Phe-Gln-Asn-Cys(Acm)-Pro-Arg-Gly-NH2.
- Vasopressin crude solution refers to the cyclized peptide in solution with all protecting groups removed with the formula:
- Vasopressin drug substance refers to the lyophilized final drug substance H-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2 Acetate salt
- the present invention relates to a method of preparing high purity vasopressin with a first step of providing protected linear resin bound vasopressin precursors, comprising cysteines with thiol residues protected with non-acid labile orthogonal protecting groups.
- the protected linear resin bound vasopressins preferably are prepared with solid phase peptide synthesis with Fmoc chemistry, wherein a resin is prepared, and a structure Fmoc-Linker-Resin is established, comprising steps of deprotection, coupling of Fmoc-protected amino acids and acetylation, and wherein the terminal Cys-derivative has Boc for protection of the N-a-amino group.
- Fmoc- Linker-Resin see for example RSC Advances JM Palomo pp 1-50“ Solid phase peptide synthesis: an overview focused on biologically relevant peptides”.
- amide linkers are preferred, especially a Rink amide,
- deprotection of acid labile protecting groups of the vasopressins is performed with a composition of an acid and a scavenger.
- the acid is selected to effectively cleave the vasopressins from the resin and to deprotect the acid labile protecting groups of the amino acids resulting in partially deprotecting the vasopressins, thereby obtaining an acidic solution of partially protected vasopressins.
- the composition includes TFA as an acid and water as scavenger.
- the composition consists of TFA and water, thereby minimizing the use of potentially poisonous and/or noxious scavenging chemicals that may form reactive species leading to impurities.
- the method comprises a step of neutralizing the acidic solution of the partially protected vasopressins with a water-soluble inorganic base to a pH >3.
- the inorganic base is an aqueous solution of 10 to 30% (w/v), preferably 20% (w/v) ammonium acetate.
- partially protected vasopressins in the neutralized solution are purified by applying the solution on a reversed phase chromatography column, using at least one aqueous acidic mobile phase comprising an organic solvent selected from methanol, ethanol, 2-propanol, and acetonitrile, preferably the organic solvent is ethanol and concentrating the eluted solution.
- aqueous acidic mobile phase comprising an organic solvent selected from methanol, ethanol, 2-propanol, and acetonitrile, preferably the organic solvent is ethanol and concentrating the eluted solution.
- the concentrated vasopressin solution is subjected to a at least one of a deprotecting agent and an oxidizing agent, to deprotect and oxidize the thiol residues to form the bridging disulfide moieties of cyclic nonapeptide vasopressins,
- a deprotecting agent and an oxidizing agent to deprotect and oxidize the thiol residues to form the bridging disulfide moieties of cyclic nonapeptide vasopressins
- iodine is used both as a deprotecting agent and an oxidizing agent.
- mercury salts and silver salts are used as a deprotection agent.
- the crude cyclic nonapeptides in the solution resulting from the deprotection and oxidation of Cys are purified with at least two reversed phase chromatography column steps of which at least one uses a mobile phase comprising a gradient of an organic solvent selected from methanol, ethanol, 2- propanol, acetonitrile, and an aqueous solution of an acid, preferably acetic acid.
- a mobile phase comprising a gradient of an organic solvent selected from methanol, ethanol, 2- propanol, acetonitrile, and an aqueous solution of an acid, preferably acetic acid.
- the purification of the partially protected nonapeptides comprises a first reversed phase chromatography step with a mobile phase having a gradient of ethanol-water comprising up to 30% (v/v) ethanol, preferably up to 15% (v/v) ethanol and an aqueous solution comprising 5 to 100 mM acetic acid, preferably 50 mM acetic acid; and a second reversed phase chromatography step with elution by a mobile phase of 40 to 60% (v/v) of ethanol and 10 to100 mM aqueous acetic acid, preferably 50 mM aqueous acetic acid.
- the elution of the second reversed phase chromatography is preferably either isocratic or performed with a short gradient or performed with combinations thereof.
- the purification of crude cyclic vasopressins comprises a first reversed phase chromatography step with a mobile phase having a gradient of ethanol-water comprising up to 40% (v/v) ethanol, preferably up to 20% (v/v) ethanol and an aqueous solution comprising 50 to 150 mM ammonium acetate, preferably 100 mM ammonium acetate, and 0.1 (v/v) to 1 % (v/v) acetic acid, preferably 0.5% (v/v) acetic acid:
- the method comprises a second reversed phase chromatography step with a mobile phase having a gradient of ethanol-water comprising up to 30% (v/v) ethanol, preferably up to 15% (v/v) ethanol and an aqueous solution comprising 10 to 100 mM, preferably 50 mM acetic acid.
- the purification further comprises a concentration step subsequent to the second reversed phase chromatography step, said concentration step comprises using mobile phase having a gradient of ethanol-water comprising up to 30% (v/v) ethanol, preferably up to 15% (v/v) ethanol and an aqueous solution comprising 10 to 100 mM acetic acid, preferably 50 mM acetic acid.
- concentration step comprises using mobile phase having a gradient of ethanol-water comprising up to 30% (v/v) ethanol, preferably up to 15% (v/v) ethanol and an aqueous solution comprising 10 to 100 mM acetic acid, preferably 50 mM acetic acid.
- the partially protected vasopressins have the structure H-Cys(X1 )-Tyr-Phe-Gln-Asn-Cys(X2)-Pro-Arg- Gly-NH2 wherein the orthogonal protecting groups X1 and X2, independently are selected from acetamido methyl (Acm) benzyl, 4-methoxybenzyl, tert-butyl, trimethylacetamidomethyl, phenylacetamidomethyl, and tert- butylmercapto, preferably X1 and X2 are Acm.
- the protected linear resin bound vasopressins have the structureBoc-Cys(Acm)-Tyr(tBu)-Phe-Gln(Trt)-Asn(Trt)- Cys(Acm)-Pro-Arg(Pbf)-Gly-Linker-resin.
- Boc-Cys(Acm)-OH has been employed to insert the terminal Cys.
- a suitable linker and a suitable resin can be found by skilled artisans.
- the reversed phase chromatography steps are performed on C-18 silica column.
- it in a second, preferred aspect of the invention, it relates to a method of producing the compound of Formula I
- the method comprises providing the linear protected vasopressins having the structure Boc-Cys(Acm)-Tyr(tBu)-Phe-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro- Arg(Pbf)-Gly-Linker-resin.
- the protected linear resin bound vasopressins preferably are prepared with solid phase peptide synthesis with Fmoc chemistry wherein a resin prepared and a structure Fmoc- Linker-Resin is established to a stepwise deprotection and comprising steps of deprotection, coupling of Fmoc-protected amino acids and acetylation, and wherein the terminal Cys-derivative has Boc for protection of the N-a-amino group.
- Fmoc- Linker-Resin see for example Molecular Biotechnology 2006, Vol. 33, page 239ff Muriel Amblard et al.
- amide linkers are preferred, especially a Rink amide
- the partly protect linear vasopressins are deprotected and cleaved simultaneously from the resin by employing an acidic agent comprising TFA and water, preferably the acid agent consists of TFA and water, and obtaining a solution of the nonapeptide H-Cys(Acm)-Tyr-Phe-Gln-Asn- Cys(Acm)-Pro-Arg-Gly-NH2.
- Next step of the method is neutralizing the solution of cleaved and partially deprotected linear vasopressins with a water-soluble inorganic base to a pH >3, preferably with an aqueous solution of 10 to 30% (w/v), preferably 20% (w/v) ammonium acetate;
- the neutralized solution is subjected to purification on a reversed phase chromatography column using at least one aqueous acidic mobile phase comprising ethanol, and the eluted solution is concentrated.
- the concentrated solution of H-Cys(Acm)-Tyr-Phe-Gln-Asn- Cys(Acm)-Pro-Arg-Gly-NH2 is subjected to an oxidizing agent, preferably iodine to deprotect the thiol residues of Cys and to form a disulfide bridge, thereby obtaining a solution comprising the compound of Formula I.
- the solution of oxidized nonapeptide is purified with at least two reversed phase chromatography steps using mobile phases comprising a gradient comprising ethanol and acetic acid.
- the purified fractions are collected and concentrated before isolation in a solid form of the acetate salt of the compound of Formula I.
- the isolation comprises lyophilization or spray drying.
- the purification of the solution of neutralized solution of partially protected nonpeptide comprises a first reversed phase chromatography step with a mobile phase having a gradient of ethanol-water comprising up to 30% (v/v) ethanol, preferably up to 15% (v/v) and 10 to 100 mM acetic acid, preferably 50 mM acetic acid; and a second reversed phase chromatography step with elution by a mobile phase of 40 to 60% (v/v) ethanol and 10 to100 mM aqueous acetic acid, preferably 50 mM aqueous acetic acid.
- the elution of the second reversed phase chromatography is preferably either isocratic or performed with a short gradient or performed with combinations thereof.
- the purification of the solution comprising the compound of Formula I comprises a first reversed phase chromatography step with a mobile phase having a gradient of ethanol-water comprising up to 40% (v/v) ethanol, preferably up to 20% (v/v) ethanol and an aqueous solution comprising 50 to 150 mM ammonium acetate, preferably 100 mM ammonium acetate, and 0.1 to 1% (v/v), preferably 0.5% (v/v) acetic acid.
- the purification comprises a concentration step subsequent to the second reversed phase chromatography step, said concentration step comprises using mobile phase having a gradient of ethanol-water comprising up to 30% (v/v) ethanol, preferably up to 15% (v/v) ethanol and an aqueous solution comprising 10 to 100 mM acetic acid, preferably 50 mM acetic acid.
- the outlined reversed phase chromatography steps are performed on C18 silica columns.
- the invention relates to vasopressin with a purity of 99.8% as obtained or obtainable by the methods as previously outlined.
- the vasopressin comprises less than 0.03% of 1 ,6’, 1’6 antiparallel dimer vasopressin and/or less than 0.03% of 1 ,1 ’,6,6’ parallel dimer of vasopressin.
- vasopressin with a purity of 99.8%, obtained or obtainable by the methods as earlier outlined and a pharmaceutically acceptable carrier.
- Fig. 1 is a schematic overview of the complete manufacturing process of vasopressin drug substance.
- Fig. 2 is a flow chart of the solid phase synthesis.
- Figs. 3A and 3B are together a schematic overview of the purification processes, the deprotection/oxidation process, concentration processes and a lyophilization process.
- the peptide is then purified by RPC and the Acm protection groups are removed by iodine oxidation with simultaneous formation of a disulphide bridge between the two Cysteine-groups of the molecule.
- the resulting solution containing crude vasopressin is used as such in the proceeding purification by RPC.
- the peptide is concentrated, filtered and finally lyophilized yielding vasopressin drug substance.
- Table 1 below outlines reagents/solvents used in the SPPS cycles for attachment of Fmoc-AA-OH/Boc-AA-OH.
- AM-resin is added to an SPPS reactor and swelled in DMF.
- the swelled AM-resin is pretreated using piperidine (20%, v/v) in DMF and then washed with DMF.
- the link between the resin and the peptide is established by a Rink type amide linker, Fmoc-Rink-OH.
- the amide linker is dissolved in HOBt and DMF and pre-activated by adding DIC.
- the preactivated amide linker and the swelled resin are mixed and NMM is added.
- the reaction mixture is stirred at ambient temperature to complete the coupling of the linker to the resin.
- the Kaiser test is performed to check the completeness of the coupling reaction. Thereafter, acetylation with AC2O and NMM is carried out and the Fmoc-Rink-AM-resin is washed with DMF.
- the sequential addition of the amino acids constituting the structure of vasopressin is accomplished by repeated cycles of deprotection, coupling (if necessary also re-coupling), acetylation and washing.
- the Fmoc group is used for temporary protection of N-a-amino groups.
- the Boc group is used for protection of the N-a-amino group.
- Removal of the Fmoc groups is achieved by treatment of the peptide resin with piperidine (20%, v/v) in DMF. Chain elongation is performed by coupling an excess of amino acid derivatives to the peptide resin as preactivated HOBt-esters in DMF. Preactivation of amino acid is done at ambient temperature for a period of time (1 -45 min), in the presence of HOBt, DMF, and DIC. Preactivation solution is added to the resin and thereafter NMM is also added.
- the peptide resin is washed with DMF and is then ready for a new synthesis cycle. After complete solid phase synthesis, the peptide resin is washed exhaustively with DMF and finally with isopropanol and dried.
- H(1-9)NH2 vasopressin neutralized solution is filtered and applied to a preparative RPC column packed with silica based stationary phase, the column is pre-equilibrated with 50 mM aqueous acetic acid.
- the adsorbed peptide is washed with 100 mM aqueous ammonium acetate followed by wash with 50 mM aqueous acetic acid and then eluted from the column using a gradient of 0% (v/v) ethanol/100% (v/v) 50 mM aqueous acetic acid to 15% (v/v) ethanol/85% (v/v) 50 mM aqueous acetic acid.
- the elution is monitored by measuring the absorbance at -280 nm.
- the fractions obtained are analyzed by analytical RP-HPLC and combined in such a way that the purity of the pool after RPC 1 is > 98 % and each individual impurity ⁇ 1.0 % and impurity D-Cys 6 -Di-Acm-vasopressin ⁇ 0.5 %.
- the pool obtained in the RPC 1 is applied to a preparative RPC column packed with silica based Cis stationary phase, pre-equilibrated with 50 mM acetic acid.
- the adsorbed peptide is washed with 50 mM aqueous acetic acid and then eluted from the column using ethanol in 50 mM aqueous acetic acid (isocratic elution).
- the elution is monitored by measuring the absorbance at -280 nm.
- the peptide solution is then concentrated to an oil.
- vasopressin crude solution obtained in oxidation step is diluted with 0.5 M ammonium acetate in 2.5% (v/v) aqueous acetic acid.
- the RPC column, packed with silica based C18 stationary phase, is equilibrated with 0.5 M ammonium acetate in 2.5% (v/v) aqueous acetic acid.
- the solution containing vasopressin is applied and the adsorbed peptide is washed with the equilibration buffers: 0.5 M ammonium acetate in 2.5% (v/v) aqueous acetic acid and 100 mM ammonium acetate in 0.5% (v/v) aqueous acetic acid, and then eluted from the column using a gradient of 0% (v/v) ethanol/100% 100 mM ammonium acetate in 0.5% (v/v) aqueous acetic acid to 20% (v/v) ethanol/80% (v/v) 100 mM ammonium acetate in 0.5% (v/v) aqueous acetic acid.
- the elution is monitored by measuring the absorbance at ⁇ 280 nm.
- the fractions obtained are analyzed by analytical HPLC and combined in such a way that the purity of the pool after RPC 3 is > 98 % and each individual impurity ⁇ 0,8 %.
- the pool obtained in the previous step is diluted with purified water.
- the RPC column, packed with silica based Cis stationary phase, is equilibrated with 50 mM aqueous acetic acid.
- the solution containing vasopressin is applied and the adsorbed peptide is washed with 50 mM aqueous acetic acid and then eluted from the column using a gradient of 0% (v/v) ethanol/100% (v/v) 50 mM aqueous acetic acid to 15% (v/v) ethanol/85% (v/v) 50 mM aqueous acetic acid.
- the elution is monitored by measuring the absorbance at ⁇ 280 nm.
- the fractions obtained are analyzed by analytical HPLC and combined in such a way that the purity of the pool after RPC 4 is > 98,0 % and each individual impurity ⁇ 0,5 %.
- the pool obtained in the RPC 4 is applied to a preparative RPC column packed with silica based Cis stationary phase, pre-equilibrated with 50 mM aqueous acetic acid as ⁇ 12 g/L CV.
- the adsorbed peptide is washed with 50 mM aqueous acetic acid and then eluted from the column using a gradient of 0% (v/v) ethanol/100% (v/v) 50 mM aqueous acetic acid to 15% (v/v) ethanol/85% (v/v) 50 mM aqueous acetic acid.
- the elution is monitored by measuring the absorbance at -280 nm.
- the fractions obtained are analyzed by analytical HPLC and combined in such a way that the purity of the pool of RPC 5 is > 98,0 %.
- the pool of purified vasopressin Prior to lyophilization, in order to reduce the amount of ethanol in the peptide solution, the pool of purified vasopressin is concentrated by evaporation to approximately 30% of starting the volume. The solution is diluted to 20-30 g/L and then filtered through a 0.45/0.2 pm membrane before the peptide solution is lyophilized.
- the time required for freeze-drying of vasopressin is at least 74 hrs.
- Lyophilized vasopressin drug substance is analyzed by analytical HPLC showing an average purity of 99.8% from seven batches having a purity ranging from 99.73 to 99.88% and less than 0.03% of 1 ,6’,1’6 antiparallel dimer vasopressin and less than 0.03% of 1 ,1 ’,6,6’ parallel dimer of vasopressin.
- Vasopressin drug substance As a representative but not limiting example, the quantities used for the manufacturing of Vasopressin drug substance are listed below. The following quantities of starting material and chemicals, used for the synthesis of Boc(1- 9)resin vasopressin (31 .2 g) are shown in Tables 2 to 4 below.
- H(1 -9)NH2 vasopressin 5g obtained from 20 g of Boc(1 -9)resin vasopressin obtained from 3.1 g of AM-resin.
- Vasopressin acetate salt from 5 g of H(1 -9)NH2 vasopressin. MWvaso P ressinx2HOAc 1148.3
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Abstract
Methods of producing vasopressin are disclosed. The methods are based on solid phase peptide synthesis, partial deprotection and resin cleavage, purification, oxidation to a cyclic crude peptide and subsequent purification steps before isolation with high purity. Also disclosed are high purity vasopressins as obtained by the methods.
Description
METHOD OF PREPARING VASOPRESSIN
Technical field
[0001] The present invention relates to a new method of preparing vasopressin.
Background of the invention
[0002] The vasopressins are hormone peptides consisting of nine amino acids (nonapeptides) and has a general function of regulating the tonicity of body fluids and control water reabsorption. The amino acid sequence of human vasopressin (antidiuretic hormone (ADH)) or arginine vasopressin (argipressin or AVP), is Cys- Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2, with the cysteine residues forming a disulfide bond and the C-terminus of the sequence converted to a primary amide. Lysine vasopressin (lypressin) has a lysine in place of the arginine as the eighth amino acid, and is found in pigs and some related animals. There are numerous previous publications disclosing processes or process steps useful to manufacture vasopressin or other disulfide bonded peptides with solid phase peptide synthesis (SPPS) as well as aligned purification steps. Some recently published patent publications exemplify and represent the state of art in vasopressin production: CN103980351 B; CN106749540B; CN106749541 B; CN106518975B;
CN106632615B; CN106699851 B; CN106518978B; CN109942686A; CN1 10016071 B; CN110003313B; CN110016070A; CN109929011 B; CN1 10078795A; W02020224580; IN201841030341 ; W02020212477; CN106866744B; CN109836470A; and IN201841035862A. In addition WO 2006/041945 discloses a method of producing vasopressin with Fmoc based SPPS using a two-step reaction to cleave and deprotect the resin bound vasopressin precursor nonapeptide and a following complicated and consuming work-up process with ether precipitation, filtration, drying, dissolution before purification of linear vasopressin precursors.
[0003] It is obvious that there is a need for improved large-scale manufacturing methods of vasopressin that combine simplification and environmental considerations with high product purity.
General description of the invention
[0004] It is an object of the present invention to provide simplified methods of large-scale production of vasopressin from Fmoc solid phase peptide synthesis with a sufficient yield and high peptide purity indicating negligible amounts of polymeric impurities, while avoiding process steps that involves evaporation and handling of hazardous or noxious solvents.
[0005] It is an another object of the present invention to provide a simple, effective cleavage and deprotection of a resin bound vasopressin precursor without using scavengers and provide a simple process for direct purification of a linear partially protected vasopressin precursor that reduces the bulk of the process related impurities formed in the SPPS and the cleavage step of linear protected vasopressins from resin.
[0006] In the following sections, chemical symbols for amino acid derivatives and peptides follow the IUPAC-IUB joint Commission on Nomenclature (see Biochem. J. 219, 345-373, 1984). The following abbreviations are used herein:
AA amino acid(s)
Acm acetamidomethyl
Boc tert-butyloxycarbonyl
DIC /V,/V-diisopropylcarbodiimide
DMF /V,/V-dimethylformamide
Fmoc 9-fluorenylmethoxycarbonyl
HOBt 1 -hydroxybenzotriazole
MeOH methanol
MS mass spectrometry
NMM /V-methylmorpholine
HPLC high performance liquid chromatography
RPC reversed phase chromatography
SPPS solid phase peptide synthesis
TFA trifluoroacetic acid
Trt trityl
[0007] As used herein, the term "orthogonal protecting group" refers to a protecting group of an amino acid side chain that is chemically resistant under one
set of selected conditions but is liable under another set of conditions. Orthogonal protecting groups for thiol containing residues include, but are not limited to, at least one of acetamido methyl (Acm), benzyl (Bzl), 4-methoxybenzyl (Mob), tertbutyl (Tbu or t-Bu), trimethylacetamidomethyl (Tacm), phenylacetamidomethyl (Phacm), or tert-butylmercapto (StBu). Preferably, the orthogonal protecting group is a non-acid labile group such as acetamido methyl. Thus, in the process of the present invention, the peptide chain is not completely deprotected after deprotection or cleavage but carries protecting groups attached to the thiol containing residues through a first purification step.
[0008] The term “scavengers” relates to organic compounds added to the cleavage and deprotection step of resin bound protected peptide to quench formed reactive ions, especially cations during this process step. Such compounds include silane scavengers such as triisopropylsilane and thiol scavengers such as dithiothreitol (DTT), anisole, thioanisole or 1 ,2-ethanedithiol. Free from scavengers in the context of the present context means free from this type of organic scavengers that may form impurities difficult to remove with following purification steps.
The following definitions are used herein:
[0009] Boc(1-9)resin vasopressin is the protected linear peptide, not cleaved from the resin: Boc-Cys(Acm)-Tyr(tBu)-Phe-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro- Arg(Pbf)-Gly-Rink-AM-resin.
[0010] H(1-9)NH2 vasopressin neutralized solution is the neutralized peptide, cleaved from the resin and partly deprotected in solution, not purified: H- Cys(Acm)-Tyr-Phe-Gln-Asn-Cys(Acm)-Pro-Arg-Gly-NH2.
[0011] Vasopressin crude solution refers to the cyclized peptide in solution with all protecting groups removed with the formula:
H-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2
[0012] Vasopressin drug substance refers to the lyophilized final drug substance
H-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2 Acetate salt
[0013] In a first aspect, the present invention relates to a method of preparing high purity vasopressin with a first step of providing protected linear resin bound vasopressin precursors, comprising cysteines with thiol residues protected with non-acid labile orthogonal protecting groups.
[0014] The protected linear resin bound vasopressins, preferably are prepared with solid phase peptide synthesis with Fmoc chemistry, wherein a resin is prepared, and a structure Fmoc-Linker-Resin is established, comprising steps of deprotection, coupling of Fmoc-protected amino acids and acetylation, and wherein the terminal Cys-derivative has Boc for protection of the N-a-amino group. There are several alternatives for the skilled person to select the construct Fmoc- Linker-Resin, see for example RSC Advances JM Palomo pp 1-50“ Solid phase peptide synthesis: an overview focused on biologically relevant peptides”. In embodiments, amide linkers are preferred, especially a Rink amide,
[0015] According to the inventive method, deprotection of acid labile protecting groups of the vasopressins is performed with a composition of an acid and a scavenger. The acid is selected to effectively cleave the vasopressins from the resin and to deprotect the acid labile protecting groups of the amino acids resulting in partially deprotecting the vasopressins, thereby obtaining an acidic solution of partially protected vasopressins. In embodiments, the composition includes TFA as an acid and water as scavenger. In preferred embodiments, the composition consists of TFA and water, thereby minimizing the use of potentially poisonous and/or noxious scavenging chemicals that may form reactive species leading to impurities.
[0016] Following cleavage/partial deprotection of the vasopressins, the method comprises a step of neutralizing the acidic solution of the partially protected vasopressins with a water-soluble inorganic base to a pH >3. In embodiments, the inorganic base is an aqueous solution of 10 to 30% (w/v), preferably 20% (w/v) ammonium acetate.
[0017] As a next step of the method, partially protected vasopressins in the neutralized solution are purified by applying the solution on a reversed phase chromatography column, using at least one aqueous acidic mobile phase comprising an organic solvent selected from methanol, ethanol, 2-propanol, and acetonitrile, preferably the organic solvent is ethanol and concentrating the eluted solution.
[0018] Following the previous purification step, the concentrated vasopressin solution is subjected to a at least one of a deprotecting agent and an oxidizing agent, to deprotect and oxidize the thiol residues to form the bridging disulfide moieties of cyclic nonapeptide vasopressins, In one embodiment, iodine is used both as a deprotecting agent and an oxidizing agent. In embodiments mercury salts and silver salts are used as a deprotection agent.
[0019] The crude cyclic nonapeptides in the solution resulting from the deprotection and oxidation of Cys are purified with at least two reversed phase chromatography column steps of which at least one uses a mobile phase comprising a gradient of an organic solvent selected from methanol, ethanol, 2- propanol, acetonitrile, and an aqueous solution of an acid, preferably acetic acid.
[0020] As a next step of the method, purified fractions are collected and concentrated before isolating the cyclic vasopressins to a product with a purity of 99.73% to 99.88%, on average 99.8%, as measured by analytical HPLC.
[0021] In embodiments of the method as outlined, the purification of the partially protected nonapeptides comprises a first reversed phase chromatography step with a mobile phase having a gradient of ethanol-water comprising up to 30% (v/v) ethanol, preferably up to 15% (v/v) ethanol and an aqueous solution comprising 5 to 100 mM acetic acid, preferably 50 mM acetic acid; and a second reversed phase chromatography step with elution by a mobile phase of 40 to 60% (v/v) of ethanol and 10 to100 mM aqueous acetic acid, preferably 50 mM aqueous acetic acid. The elution of the second reversed phase chromatography is preferably
either isocratic or performed with a short gradient or performed with combinations thereof.
[0022] In embodiments of the method, the purification of crude cyclic vasopressins comprises a first reversed phase chromatography step with a mobile phase having a gradient of ethanol-water comprising up to 40% (v/v) ethanol, preferably up to 20% (v/v) ethanol and an aqueous solution comprising 50 to 150 mM ammonium acetate, preferably 100 mM ammonium acetate, and 0.1 (v/v) to 1 % (v/v) acetic acid, preferably 0.5% (v/v) acetic acid: Further, according to this embodiment, the method comprises a second reversed phase chromatography step with a mobile phase having a gradient of ethanol-water comprising up to 30% (v/v) ethanol, preferably up to 15% (v/v) ethanol and an aqueous solution comprising 10 to 100 mM, preferably 50 mM acetic acid. Preferably, the purification further comprises a concentration step subsequent to the second reversed phase chromatography step, said concentration step comprises using mobile phase having a gradient of ethanol-water comprising up to 30% (v/v) ethanol, preferably up to 15% (v/v) ethanol and an aqueous solution comprising 10 to 100 mM acetic acid, preferably 50 mM acetic acid.
[0023] In embodiments of the methods as outlined, the partially protected vasopressins have the structure H-Cys(X1 )-Tyr-Phe-Gln-Asn-Cys(X2)-Pro-Arg- Gly-NH2 wherein the orthogonal protecting groups X1 and X2, independently are selected from acetamido methyl (Acm) benzyl, 4-methoxybenzyl, tert-butyl, trimethylacetamidomethyl, phenylacetamidomethyl, and tert- butylmercapto, preferably X1 and X2 are Acm.
[0024] In embodiments of the method, the protected linear resin bound vasopressins have the structureBoc-Cys(Acm)-Tyr(tBu)-Phe-Gln(Trt)-Asn(Trt)- Cys(Acm)-Pro-Arg(Pbf)-Gly-Linker-resin. In such embodiments Boc-Cys(Acm)-OH has been employed to insert the terminal Cys. A suitable linker and a suitable resin can be found by skilled artisans.
[0025] In useful embodiments of the method, the reversed phase chromatography steps are performed on C-18 silica column.
[0026] In a second, preferred aspect of the invention, it relates to a method of producing the compound of Formula I
Formula I
[0027]The method comprises providing the linear protected vasopressins having the structure Boc-Cys(Acm)-Tyr(tBu)-Phe-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro- Arg(Pbf)-Gly-Linker-resin. As with the first aspect of the invention, the protected linear resin bound vasopressins, preferably are prepared with solid phase peptide synthesis with Fmoc chemistry wherein a resin prepared and a structure Fmoc- Linker-Resin is established to a stepwise deprotection and comprising steps of deprotection, coupling of Fmoc-protected amino acids and acetylation, and wherein the terminal Cys-derivative has Boc for protection of the N-a-amino group. There are several alternatives for the skilled person to select the construct Fmoc- Linker-Resin, see for example Molecular Biotechnology 2006, Vol. 33, page 239ff Muriel Amblard et al. In embodiments amide linkers are preferred, especially a Rink amide,
[0028] In a first step of the method, the partly protect linear vasopressins are deprotected and cleaved simultaneously from the resin by employing an acidic agent comprising TFA and water, preferably the acid agent consists of TFA and water, and obtaining a solution of the nonapeptide H-Cys(Acm)-Tyr-Phe-Gln-Asn- Cys(Acm)-Pro-Arg-Gly-NH2.
[0029] Next step of the method is neutralizing the solution of cleaved and partially deprotected linear vasopressins with a water-soluble inorganic base to a pH >3, preferably with an aqueous solution of 10 to 30% (w/v), preferably 20% (w/v) ammonium acetate;
[0030]The neutralized solution is subjected to purification on a reversed phase chromatography column using at least one aqueous acidic mobile phase comprising ethanol, and the eluted solution is concentrated.
[0031] As next step, the concentrated solution of H-Cys(Acm)-Tyr-Phe-Gln-Asn- Cys(Acm)-Pro-Arg-Gly-NH2 is subjected to an oxidizing agent, preferably iodine to deprotect the thiol residues of Cys and to form a disulfide bridge, thereby obtaining a solution comprising the compound of Formula I.
[0032]The solution of oxidized nonapeptide is purified with at least two reversed phase chromatography steps using mobile phases comprising a gradient comprising ethanol and acetic acid. The purified fractions are collected and concentrated before isolation in a solid form of the acetate salt of the compound of Formula I. Preferably, the isolation comprises lyophilization or spray drying.
[0033] In embodiments of the method, the purification of the solution of neutralized solution of partially protected nonpeptide comprises a first reversed phase chromatography step with a mobile phase having a gradient of ethanol-water comprising up to 30% (v/v) ethanol, preferably up to 15% (v/v) and 10 to 100 mM acetic acid, preferably 50 mM acetic acid; and a second reversed phase chromatography step with elution by a mobile phase of 40 to 60% (v/v) ethanol and 10 to100 mM aqueous acetic acid, preferably 50 mM aqueous acetic acid. The elution of the second reversed phase chromatography is preferably either isocratic or performed with a short gradient or performed with combinations thereof.
[0034] In embodiments of the method, the purification of the solution comprising the compound of Formula I comprises a first reversed phase chromatography step with a mobile phase having a gradient of ethanol-water comprising up to 40% (v/v) ethanol, preferably up to 20% (v/v) ethanol and an aqueous solution comprising 50 to 150 mM ammonium acetate, preferably 100 mM ammonium acetate, and 0.1 to 1% (v/v), preferably 0.5% (v/v) acetic acid. Further to this embodiment, it comprises a second reversed phase chromatography step with a mobile phase having a gradient of ethanol-water comprising up to 30% (v/v) ethanol, preferably up to 15% (v/v) ethanol and an aqueous solution comprising 10 to 100 mM, preferably 50 mM acetic acid. In embodiments, the purification comprises a
concentration step subsequent to the second reversed phase chromatography step, said concentration step comprises using mobile phase having a gradient of ethanol-water comprising up to 30% (v/v) ethanol, preferably up to 15% (v/v) ethanol and an aqueous solution comprising 10 to 100 mM acetic acid, preferably 50 mM acetic acid.
[0035] In useful embodiments, the outlined reversed phase chromatography steps are performed on C18 silica columns.
[0036] In further aspects the invention relates to vasopressin with a purity of 99.8% as obtained or obtainable by the methods as previously outlined.
[0037] In embodiments, the vasopressin comprises less than 0.03% of 1 ,6’, 1’6 antiparallel dimer vasopressin and/or less than 0.03% of 1 ,1 ’,6,6’ parallel dimer of vasopressin.
[0038] In still further aspects of the invention, it relates to a pharmaceutical composition comprising vasopressin with a purity of 99.8%, obtained or obtainable by the methods as earlier outlined and a pharmaceutically acceptable carrier.
Detailed and experimental description
Fig. 1 is a schematic overview of the complete manufacturing process of vasopressin drug substance.
Fig. 2 is a flow chart of the solid phase synthesis.
Figs. 3A and 3B are together a schematic overview of the purification processes, the deprotection/oxidation process, concentration processes and a lyophilization process.
Introduction
[0039] In the following a manufacturing process based on Fmoc solid phase peptide synthesis is described that is developed for large-scale manufacture of vasopressin drug substance. The synthesis of Boc(1-9)resin vasopressin is
performed by solid phase peptide chemistry. The protected linear peptide is partly deprotected and cleaved from the resin by TFA/H2O, the peptide solution obtained is neutralized with aqueous ammonium acetate and filtered, yielding H(1 -9)NH2 vasopressin neutralized solution. The peptide is then purified by RPC and the Acm protection groups are removed by iodine oxidation with simultaneous formation of a disulphide bridge between the two Cysteine-groups of the molecule. The resulting solution containing crude vasopressin is used as such in the proceeding purification by RPC. The peptide is concentrated, filtered and finally lyophilized yielding vasopressin drug substance. Table 1 below outlines reagents/solvents used in the SPPS cycles for attachment of Fmoc-AA-OH/Boc-AA-OH.
Cycle Reagent/solvent Function
1 20% piperidine/DMF Deprotection (multiple)
2 DMF Wash continuously
3 Evaluation Efficiency check;
Chloranil test
4 Fmoc-AA-OH/Boc-AA- Coupling
OH, HOBt, DMF, DIC and NMM
5 Evaluation Efficiency check; Kaiser test/Chloranil test
6a Fmoc-AA-OH/Boc-AA- Recoupling (optional)
OH, HOBt, DMF, DIC and NMM
6b AC2O and NMM Acetylation
7 DMF Wash continuously
Table 1
Resin
[0040] AM-resin is added to an SPPS reactor and swelled in DMF. The swelled AM-resin is pretreated using piperidine (20%, v/v) in DMF and then washed with DMF. The link between the resin and the peptide is established by a Rink type
amide linker, Fmoc-Rink-OH. The amide linker is dissolved in HOBt and DMF and pre-activated by adding DIC. The preactivated amide linker and the swelled resin are mixed and NMM is added. The reaction mixture is stirred at ambient temperature to complete the coupling of the linker to the resin. The Kaiser test is performed to check the completeness of the coupling reaction. Thereafter, acetylation with AC2O and NMM is carried out and the Fmoc-Rink-AM-resin is washed with DMF.
Coupling of amino acids
[0041 ]The sequential addition of the amino acids constituting the structure of vasopressin is accomplished by repeated cycles of deprotection, coupling (if necessary also re-coupling), acetylation and washing. The Fmoc group is used for temporary protection of N-a-amino groups. For the terminal Cys-derivative, the Boc group is used for protection of the N-a-amino group. Removal of the Fmoc groups is achieved by treatment of the peptide resin with piperidine (20%, v/v) in DMF. Chain elongation is performed by coupling an excess of amino acid derivatives to the peptide resin as preactivated HOBt-esters in DMF. Preactivation of amino acid is done at ambient temperature for a period of time (1 -45 min), in the presence of HOBt, DMF, and DIC. Preactivation solution is added to the resin and thereafter NMM is also added.
[0042]Coupling is controlled by the Kaiser colour test, detecting residual resinbound amino groups. Where applicable, the Chloranil test is used. In the coupling reaction,
[0043] if the Kaiser test is negative, as indicated by a yellow to a yellow-greenish colour of the resin bead and by the yellow colour of the test solution, the reaction is considered to be complete and the reaction mixture is acetylated.
[0044] If the Kaiser test is positive, as indicated by a blue or green colour of the test solution, recoupling (optional) or acetylation (end capping) is performed.
[0045] Recoupling is performed by addition of preactivated amino acid derivative, after filtration.
[0046]Acetylation is performed by addition of acetic anhydride and NMM to the resin in DMF.
[0047]The peptide resin is washed with DMF and is then ready for a new synthesis cycle. After complete solid phase synthesis, the peptide resin is washed exhaustively with DMF and finally with isopropanol and dried.
Deprotection, cleavage from resin and neutralization
[0048] Deprotection (partly) and cleavage of the vasopressin peptide from the resin is performed using acidolysis, see Table 2. The protected linear peptide resin is treated with cold TFA/H2O (19:1 ) and stirred. After filtration, the peptide solution is treated (“neutralized”) with ammonium acetate/FhO (~ 20%, w/v) and pH is checked, expected to be 3.5 ±0.5. The peptide solution is then filtered yielding H(1-9)NH2 vasopressin neutralized solution.
THE PURIFICATION PROCESSES, THE DEPROTECTION/OXIDATION PROCESS, CONCENTRATION PROCESSES AND THE LYOPHILIZATION PROCESS
The process steps of Figs. 3A and 3B are described below. Any concentration given in the examples below is meant as +/- 25% of the molarity/concentration stated.
Purification of H(1-9)NH2 vasopressin neutralized solution by preparative RPC (RPC 1)
[0049]H(1-9)NH2 vasopressin neutralized solution is filtered and applied to a preparative RPC column packed with silica based stationary phase, the column is pre-equilibrated with 50 mM aqueous acetic acid. The adsorbed peptide is washed with 100 mM aqueous ammonium acetate followed by wash with 50 mM aqueous acetic acid and then eluted from the column using a gradient of 0% (v/v) ethanol/100% (v/v) 50 mM aqueous acetic acid to 15% (v/v) ethanol/85% (v/v) 50
mM aqueous acetic acid. The elution is monitored by measuring the absorbance at -280 nm.
Process control:
The fractions obtained are analyzed by analytical RP-HPLC and combined in such a way that the purity of the pool after RPC 1 is > 98 % and each individual impurity < 1.0 % and impurity D-Cys6-Di-Acm-vasopressin < 0.5 %.
Concentration by preparative RPC (RPC 2)
[0050]The pool obtained in the RPC 1 is applied to a preparative RPC column packed with silica based Cis stationary phase, pre-equilibrated with 50 mM acetic acid. The adsorbed peptide is washed with 50 mM aqueous acetic acid and then eluted from the column using ethanol in 50 mM aqueous acetic acid (isocratic elution). The elution is monitored by measuring the absorbance at -280 nm. The peptide solution is then concentrated to an oil.
Oxidation and formation of the disulfide bridge
[0051]Tri Acm protecting groups on cysteines are removed by treatment with I2 (dissolved in methanol) in acetic acid/F . On this treatment the disulfide bridge is formed simultaneously. When the reaction is completed, a solution of ascorbic acid/ammonium acetate in H2O is added to quench the reaction and destroy the excess of I2. The resulting solution, containing crude vasopressin, is used in the subsequent purification procedure.
Purification by preparative RPC (RPC 3)
[0052]The vasopressin crude solution obtained in oxidation step, is diluted with 0.5 M ammonium acetate in 2.5% (v/v) aqueous acetic acid. The RPC column, packed with silica based C18 stationary phase, is equilibrated with 0.5 M ammonium acetate in 2.5% (v/v) aqueous acetic acid. The solution containing vasopressin is applied and the adsorbed peptide is washed with the equilibration buffers: 0.5 M ammonium acetate in 2.5% (v/v) aqueous acetic acid and 100 mM ammonium acetate in 0.5% (v/v) aqueous acetic acid, and then eluted from the
column using a gradient of 0% (v/v) ethanol/100% 100 mM ammonium acetate in 0.5% (v/v) aqueous acetic acid to 20% (v/v) ethanol/80% (v/v) 100 mM ammonium acetate in 0.5% (v/v) aqueous acetic acid. The elution is monitored by measuring the absorbance at ~280 nm.
Process control:
The fractions obtained are analyzed by analytical HPLC and combined in such a way that the purity of the pool after RPC 3 is > 98 % and each individual impurity < 0,8 %.
Purification by preparative RPC (RPC 4)
[0053]The pool obtained in the previous step, is diluted with purified water. The RPC column, packed with silica based Cis stationary phase, is equilibrated with 50 mM aqueous acetic acid. The solution containing vasopressin is applied and the adsorbed peptide is washed with 50 mM aqueous acetic acid and then eluted from the column using a gradient of 0% (v/v) ethanol/100% (v/v) 50 mM aqueous acetic acid to 15% (v/v) ethanol/85% (v/v) 50 mM aqueous acetic acid. The elution is monitored by measuring the absorbance at ~280 nm.
Process control:
The fractions obtained are analyzed by analytical HPLC and combined in such a way that the purity of the pool after RPC 4 is > 98,0 % and each individual impurity < 0,5 %.
Concentration by preparative RPC (RPC 5)
[0054]The pool obtained in the RPC 4 is applied to a preparative RPC column packed with silica based Cis stationary phase, pre-equilibrated with 50 mM aqueous acetic acid as < 12 g/L CV. The adsorbed peptide is washed with 50 mM aqueous acetic acid and then eluted from the column using a gradient of 0% (v/v) ethanol/100% (v/v) 50 mM aqueous acetic acid to 15% (v/v) ethanol/85% (v/v) 50
mM aqueous acetic acid. The elution is monitored by measuring the absorbance at -280 nm.
Process control:
The fractions obtained are analyzed by analytical HPLC and combined in such a way that the purity of the pool of RPC 5 is > 98,0 %.
Isolation by concentration filtration and lyophilization
[0055] Prior to lyophilization, in order to reduce the amount of ethanol in the peptide solution, the pool of purified vasopressin is concentrated by evaporation to approximately 30% of starting the volume. The solution is diluted to 20-30 g/L and then filtered through a 0.45/0.2 pm membrane before the peptide solution is lyophilized.
[0056] In the lyophilization process yielding vasopressin drug substance, the time required for freeze-drying of vasopressin is at least 74 hrs.
[0057]Primary drying is performed at 0.5 mbar.
[0058] Secondary drying is performed at < 0.05 mbar.
Purity of lyophilized vasopressin drug substance
[0059] Lyophilized vasopressin drug substance is analyzed by analytical HPLC showing an average purity of 99.8% from seven batches having a purity ranging from 99.73 to 99.88% and less than 0.03% of 1 ,6’,1’6 antiparallel dimer vasopressin and less than 0.03% of 1 ,1 ’,6,6’ parallel dimer of vasopressin.
Example 1
[0060] As a representative but not limiting example, the quantities used for the manufacturing of Vasopressin drug substance are listed below. The following quantities of starting material and chemicals, used for the synthesis of Boc(1- 9)resin vasopressin (31 .2 g) are shown in Tables 2 to 4 below.
Table 2
Chemical Purpose Quantity
Acetic anhydride Reagent 140 ml
Amide linker / Fmoc-Rink-OH Linker 11 g
AM-resin The solid phase for 5 g (2-4 mmol/g) peptide synthesis
Isopropanol Solvent 0.8 kg
/V,/V-Diisopropylcarbodiimide Reagent 31 ml
/V,/V-Dimethylformamide Solvent 12,5 kg
/V-Methylmorpholine Catalyst/base 11 ml
Piperidine Reagent 190 ml
Table 3
[0061]
[0062]in and neutralization, used for the synthesis of H(1-9)NH2 vasopressin neutralized solution from 20 g Boc(1-9)resin vasopressin, are shown below in Table 4.
[0063]
Chemical Purpose Quantity
Ammonium acetate Neutralization 0.2 kg
Purified water Scavenger 10 ml
Purified water Solvent 11 ml
Trifluoroacetic acid Reagent 0.3 kg
Table 4
[0039] Chemicals used for purification, deprotection/oxidation and purification in the synthesis of 2.9 g vasopressin drug substance from 5 g H(1-9)NH2 vasopressin in neutralized solution, obtained from 20 g Boc(1-9)resin vasopressin, are shown In Table 5.
Chemical Purpose Quantity
Acetic acid Reagent/solvent 0.7 L
Ammonium acetate Reagent 0.5 kg
Ascorbic acid Reagent 0.02 kg
Ethanol Reagent/solvent 7 L
Iodine Reagent 21 g
Methanol Solvent 0.2 L
Purified water Solvent 50 L
Table 5
[0040] The yields from manufacturing of vasopressin drug substance according to presently invented method, as described above, are summarized in Table 6.
1 ) Theoretical value of synthesized Boc(1 -9) vasopressin bound to 3.1 g AM-resin
2) H(1 -9)NH2 vasopressin, 5g obtained from 20 g of Boc(1 -9)resin vasopressin obtained from 3.1 g of AM-resin. MWH(I-9)NH2 vasopressin — 1170.4
3) Vasopressin acetate salt from 5 g of H(1 -9)NH2 vasopressin. MWvasoPressinx2HOAc = 1148.3
Table 6
Claims
1 . A method of producing vasopressin of Formula I
H-Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2
I _ I
Formula I with a purity of 99.8%, comprising: a) providing the vasopressin precursors having the structure Boc-Cys(Acm)- Tyr(tBu)-Phe-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-Arg(Pbf)-Gly-Linker-resin; b) partly deprotecting and cleaving the linear vasopressin precursors from the resin with agent comprising a strong acid and is free from organic scavengers, preferably the agent consists of TFA and water, and obtaining a solution of the partially protected vasopressins having the formula H-Cys(Acm)-Tyr-Phe-Gln- Asn-Cys(Acm)-Pro-Arg-Gly-NH2; c) neutralizing the solution obtained from step b) with a water-soluble inorganic base to a pH >3, preferably with an aqueous solution comprising 10 to 30% (w/v), preferably 20% (w/v) ammonium acetate; d) purifying the neutralized solution directly obtained from step c) with one or more reversed phase chromatography columns using at least one aqueous acidic mobile phase comprising ethanol, and concentrating the eluted solution; e) subjecting the concentrated solution of H-Cys(Acm)-Tyr-Phe-Gln-Asn- Cys(Acm)-Pro-Arg-Gly-NH2 from step d) to an oxidizing agent, preferably iodine to deprotect the thiol residues of Cys and to form disulfide bridges, thereby obtaining a solution comprising the compound of Formula I; f) purifying the oxidized vasopressin in the solution from step e) with at least two reversed phase chromatography steps using mobile phases, comprising a gradient comprising ethanol and acetic acid; and g) collecting and concentrating purified fractions obtained from step f); and
h) isolating from the resulting solution of step g) by lyophilization a solid form of a salt of the compound of Formula I at a purity of 99.8% and less than 0.03% of 1 ,6’,T6 antiparallel dimer vasopressin and less than 0.03% of 1 , 1 ’,6,6’ parallel dimer of vasopressin.
2. The method according to claim 1 , wherein the purification step d) comprises:
(i) a first reversed chromatography step with a mobile phase having a gradient of ethanol-water comprising up to 30% (v/v) ethanol, preferably up to 15% (v/v) and 10 to 100 mM aqueous acetic acid, preferably 50 mM aqueous acetic acid; and
(ii) a second reversed chromatography step with elution by a mobile phase of 40 to 60% (v/v) ethanol and 10 to100 mM aqueous acetic acid, preferably 50 mM aqueous acetic acid.
3. The method according to claim 1 or 2, wherein the purification in step f) comprises:
(i) a first reversed chromatography step with a mobile phase having a gradient of ethanol-water comprising up to 40% (v/v) ethanol, preferably up to 20% (v/v) ethanol and an aqueous solution comprising 50 to 150 mM ammonium acetate, preferably 100 mM ammonium acetate, and 0.1 to 1 % (v/v), preferably 0.5% (v/v) acetic acid;
(ii) a second reversed chromatography step with a mobile phase having a gradient of ethanol-water comprising up to 30% (v/v) ethanol, preferably up to 15% (v/v) ethanol and an aqueous solution comprising 10 to 100 mM, preferably 50 mM acetic acid.
4. The method according to claim 3, wherein the purification step f) further comprises a concentration step subsequent to the second reversed chromatography step, said concentration step comprises using a mobile phase having a gradient of ethanol-water comprising up to 30% (v/v) ethanol, preferably up to 15% (v/v) ethanol and an aqueous solution comprising and 10 to 100 mM acetic acid, preferably 50 mM acetic acid.
The method according to any one of claims 1 to 4, wherein the reversed chromatography steps are performed on C18 silica columns. The method according to any one of claims 1 to 5, wherein acetate salt of vasopressin is formed. An isolated vasopressin obtained or obtainable by method according to any one of claims 1 to 5 with a purity of 99.8%. The isolated vasopressin according to claim 7, comprising less than 0.03% of 1 ,6’,1’6 antiparallel dimer vasopressin and less than 0.03% of 1 , 1 ’,6,6’ parallel dimer of vasopressin. The isolated vasopressin according to claim 7 or 8 in lyophilized form. A pharmaceutical composition, comprising vasopressin according to any one of claims 7 to 9 and a pharmaceutically acceptable carrier.
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WO2006041945A2 (en) * | 2004-10-04 | 2006-04-20 | Novetide, Ltd. | A counterion exchange process for peptides |
EP1511761B1 (en) * | 2003-04-07 | 2006-08-02 | Novetide Ltd. | Process for production of cyclic peptides |
WO2006119388A2 (en) * | 2005-05-03 | 2006-11-09 | Novetide, Ltd. | Methods for the production of peptide having a c-terminal amide |
WO2010089757A2 (en) * | 2008-11-07 | 2010-08-12 | Usv Limited | An improved process for synthesis of cyclic octapeptide |
US20120094910A1 (en) * | 2009-04-06 | 2012-04-19 | Ananda Kuppanna | Improved process for the preparation of desmopressin or its pharmaceutically acceptable salts |
WO2015152565A1 (en) * | 2014-03-31 | 2015-10-08 | 애니젠 주식회사 | Method for preparing desmopressin |
US20210087226A1 (en) * | 2019-05-06 | 2021-03-25 | Sph No.1 Biochemical & Pharmaceutical Co., Ltd. | Method for refining vasopressin |
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EP1511761B1 (en) * | 2003-04-07 | 2006-08-02 | Novetide Ltd. | Process for production of cyclic peptides |
WO2006041945A2 (en) * | 2004-10-04 | 2006-04-20 | Novetide, Ltd. | A counterion exchange process for peptides |
WO2006119388A2 (en) * | 2005-05-03 | 2006-11-09 | Novetide, Ltd. | Methods for the production of peptide having a c-terminal amide |
WO2010089757A2 (en) * | 2008-11-07 | 2010-08-12 | Usv Limited | An improved process for synthesis of cyclic octapeptide |
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