EP4181946A1 - Verbessertes reinigungsverfahren für semaglutid - Google Patents
Verbessertes reinigungsverfahren für semaglutidInfo
- Publication number
- EP4181946A1 EP4181946A1 EP21846746.2A EP21846746A EP4181946A1 EP 4181946 A1 EP4181946 A1 EP 4181946A1 EP 21846746 A EP21846746 A EP 21846746A EP 4181946 A1 EP4181946 A1 EP 4181946A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- semaglutide
- phosphate
- sodium
- purification
- buffer
- 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.)
- Pending
Links
- DLSWIYLPEUIQAV-UHFFFAOYSA-N Semaglutide Chemical compound CCC(C)C(NC(=O)C(Cc1ccccc1)NC(=O)C(CCC(O)=O)NC(=O)C(CCCCNC(=O)COCCOCCNC(=O)COCCOCCNC(=O)CCC(NC(=O)CCCCCCCCCCCCCCCCC(O)=O)C(O)=O)NC(=O)C(C)NC(=O)C(C)NC(=O)C(CCC(N)=O)NC(=O)CNC(=O)C(CCC(O)=O)NC(=O)C(CC(C)C)NC(=O)C(Cc1ccc(O)cc1)NC(=O)C(CO)NC(=O)C(CO)NC(=O)C(NC(=O)C(CC(O)=O)NC(=O)C(CO)NC(=O)C(NC(=O)C(Cc1ccccc1)NC(=O)C(NC(=O)CNC(=O)C(CCC(O)=O)NC(=O)C(C)(C)NC(=O)C(N)Cc1cnc[nH]1)C(C)O)C(C)O)C(C)C)C(=O)NC(C)C(=O)NC(Cc1c[nH]c2ccccc12)C(=O)NC(CC(C)C)C(=O)NC(C(C)C)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CCCNC(N)=N)C(=O)NCC(O)=O DLSWIYLPEUIQAV-UHFFFAOYSA-N 0.000 title claims abstract description 109
- 108010060325 semaglutide Proteins 0.000 title claims abstract description 109
- 229950011186 semaglutide Drugs 0.000 title claims abstract description 106
- 238000000746 purification Methods 0.000 title claims abstract description 49
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 19
- 239000010452 phosphate Substances 0.000 claims abstract description 19
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 19
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims abstract description 15
- 229910052708 sodium Inorganic materials 0.000 claims abstract description 15
- 239000011734 sodium Substances 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims description 33
- 239000000203 mixture Substances 0.000 claims description 24
- 239000000872 buffer Substances 0.000 claims description 21
- 238000004007 reversed phase HPLC Methods 0.000 claims description 20
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 20
- 239000003960 organic solvent Substances 0.000 claims description 18
- 235000021317 phosphate Nutrition 0.000 claims description 18
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 15
- 230000005526 G1 to G0 transition Effects 0.000 claims description 14
- 235000015424 sodium Nutrition 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 13
- 239000008363 phosphate buffer Substances 0.000 claims description 11
- 230000002378 acidificating effect Effects 0.000 claims description 10
- 238000011097 chromatography purification Methods 0.000 claims description 9
- 238000010828 elution Methods 0.000 claims description 9
- 239000001488 sodium phosphate Substances 0.000 claims description 9
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 9
- 235000011008 sodium phosphates Nutrition 0.000 claims description 9
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 claims description 9
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 239000001099 ammonium carbonate Substances 0.000 claims description 8
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 7
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical group OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 7
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 7
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 7
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 7
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical group [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 7
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 claims description 6
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 6
- 235000012538 ammonium bicarbonate Nutrition 0.000 claims description 6
- 235000002639 sodium chloride Nutrition 0.000 claims description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 5
- 235000011007 phosphoric acid Nutrition 0.000 claims description 5
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 4
- 238000002955 isolation Methods 0.000 claims description 4
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 4
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 4
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 4
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 claims description 4
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 3
- 239000004254 Ammonium phosphate Substances 0.000 claims description 3
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical class C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 claims description 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims description 3
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 3
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 3
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 3
- 235000019270 ammonium chloride Nutrition 0.000 claims description 3
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 3
- 229910000148 ammonium phosphate Inorganic materials 0.000 claims description 3
- 235000019289 ammonium phosphates Nutrition 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 239000001166 ammonium sulphate Substances 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 150000001768 cations Chemical class 0.000 claims description 3
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 3
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 235000013922 glutamic acid Nutrition 0.000 claims description 3
- 239000004220 glutamic acid Substances 0.000 claims description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 3
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 3
- 229910000160 potassium phosphate Inorganic materials 0.000 claims description 3
- 235000011009 potassium phosphates Nutrition 0.000 claims description 3
- 125000002924 primary amino group Chemical class [H]N([H])* 0.000 claims description 3
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 3
- 235000011152 sodium sulphate Nutrition 0.000 claims description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 3
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 2
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 claims description 2
- 235000012501 ammonium carbonate Nutrition 0.000 claims description 2
- 239000000908 ammonium hydroxide Substances 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 235000011056 potassium acetate Nutrition 0.000 claims description 2
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 2
- 235000011181 potassium carbonates Nutrition 0.000 claims description 2
- 239000001632 sodium acetate Substances 0.000 claims description 2
- 235000017281 sodium acetate Nutrition 0.000 claims description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 2
- 235000017550 sodium carbonate Nutrition 0.000 claims description 2
- 239000011260 aqueous acid Substances 0.000 claims 4
- 239000008194 pharmaceutical composition Substances 0.000 claims 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims 3
- 239000000546 pharmaceutical excipient Substances 0.000 claims 3
- 150000003839 salts Chemical class 0.000 claims 3
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims 2
- 150000001450 anions Chemical class 0.000 claims 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims 1
- 239000005695 Ammonium acetate Substances 0.000 claims 1
- 230000002776 aggregation Effects 0.000 claims 1
- 238000004220 aggregation Methods 0.000 claims 1
- 235000019257 ammonium acetate Nutrition 0.000 claims 1
- 229940043376 ammonium acetate Drugs 0.000 claims 1
- 239000012296 anti-solvent Substances 0.000 claims 1
- 239000012062 aqueous buffer Substances 0.000 claims 1
- 235000003704 aspartic acid Nutrition 0.000 claims 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims 1
- 206010061592 cardiac fibrillation Diseases 0.000 claims 1
- 239000003937 drug carrier Substances 0.000 claims 1
- 230000002600 fibrillogenic effect Effects 0.000 claims 1
- 235000019253 formic acid Nutrition 0.000 claims 1
- 238000001879 gelation Methods 0.000 claims 1
- 235000014304 histidine Nutrition 0.000 claims 1
- 229910017053 inorganic salt Inorganic materials 0.000 claims 1
- 150000007524 organic acids Chemical class 0.000 claims 1
- 235000008729 phenylalanine Nutrition 0.000 claims 1
- 239000001103 potassium chloride Substances 0.000 claims 1
- 235000011164 potassium chloride Nutrition 0.000 claims 1
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims 1
- 229910052939 potassium sulfate Inorganic materials 0.000 claims 1
- 239000001120 potassium sulphate Substances 0.000 claims 1
- 235000011151 potassium sulphates Nutrition 0.000 claims 1
- 238000001556 precipitation Methods 0.000 claims 1
- 235000002374 tyrosine Nutrition 0.000 claims 1
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 60
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 49
- 239000012071 phase Substances 0.000 description 40
- 108090000765 processed proteins & peptides Proteins 0.000 description 24
- 239000000377 silicon dioxide Substances 0.000 description 22
- 125000001183 hydrocarbyl group Chemical group 0.000 description 17
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- 239000007788 liquid Substances 0.000 description 9
- 238000010647 peptide synthesis reaction Methods 0.000 description 7
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 150000001298 alcohols Chemical class 0.000 description 5
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- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 4
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- FUOOLUPWFVMBKG-UHFFFAOYSA-N 2-Aminoisobutyric acid Chemical compound CC(C)(N)C(O)=O FUOOLUPWFVMBKG-UHFFFAOYSA-N 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
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- DDYAPMZTJAYBOF-ZMYDTDHYSA-N (3S)-4-[[(2S)-1-[[(2S)-1-[[(2S)-5-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-4-amino-1-[[(2S,3R)-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-4-amino-1-[[(2S)-1-[[(2S)-4-amino-1-[[(2S)-4-amino-1-[[(2S,3S)-1-[[(1S)-1-carboxyethyl]amino]-3-methyl-1-oxopentan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-1-oxohexan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-4-methylsulfanyl-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-1,5-dioxopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S,3R)-2-[[2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-amino-3-(1H-imidazol-4-yl)propanoyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]acetyl]amino]-3-hydroxybutanoyl]amino]-3-phenylpropanoyl]amino]-3-hydroxybutanoyl]amino]-3-hydroxypropanoyl]amino]-3-carboxypropanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-3-hydroxypropanoyl]amino]hexanoyl]amino]-3-(4-hydroxyphenyl)propanoyl]amino]-4-methylpentanoyl]amino]-3-carboxypropanoyl]amino]-3-hydroxypropanoyl]amino]-5-carbamimidamidopentanoyl]amino]-5-carbamimidamidopentanoyl]amino]propanoyl]amino]-5-oxopentanoyl]amino]-4-oxobutanoic acid Chemical class [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O DDYAPMZTJAYBOF-ZMYDTDHYSA-N 0.000 description 2
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- 108010088406 Glucagon-Like Peptides Proteins 0.000 description 2
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- 238000010521 absorption reaction Methods 0.000 description 2
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- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
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- 238000007086 side reaction Methods 0.000 description 2
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- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000000825 ultraviolet detection Methods 0.000 description 2
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 1
- SNRUBQQJIBEYMU-UHFFFAOYSA-N Dodecane Chemical group CCCCCCCCCCCC SNRUBQQJIBEYMU-UHFFFAOYSA-N 0.000 description 1
- 101710198884 GATA-type zinc finger protein 1 Proteins 0.000 description 1
- 102000051325 Glucagon Human genes 0.000 description 1
- 108060003199 Glucagon Proteins 0.000 description 1
- 102000007446 Glucagon-Like Peptide-1 Receptor Human genes 0.000 description 1
- 108010086246 Glucagon-Like Peptide-1 Receptor Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 101000788682 Homo sapiens GATA-type zinc finger protein 1 Proteins 0.000 description 1
- 102100040918 Pro-glucagon Human genes 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 125000000738 acetamido group Chemical group [H]C([H])([H])C(=O)N([H])[*] 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000005341 cation exchange Methods 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000006482 condensation reaction Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 230000006240 deamidation Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 125000003438 dodecyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 229940088679 drug related substance Drugs 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 description 1
- 229960004666 glucagon Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000002641 glycemic effect Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229940025708 injectable product Drugs 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000013178 mathematical model Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000010979 pH adjustment Methods 0.000 description 1
- 230000007030 peptide scission Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 125000000025 triisopropylsilyl group Chemical group C(C)(C)[Si](C(C)C)(C(C)C)* 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/605—Glucagons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present application relates to improved and effective purification processes for semaglutide.
- the present invention is also related to stable semaglutide.
- the present invention is also related to stable semaglutide comprising phosphate content in the range between 5% to 15% w/w and sodium content in the range between about 4% to about 10% w/w.
- Semaglutide is a GLP-1 analogue with 94% sequence homology to human GLP-1.
- GLP-1 is a physiological hormone that has multiple actions on glucose, mediated by the GLP-1 receptors.
- Semaglutide proprietary name OZEMPIC ® , developed by Novo Nordisk and first approved by USFDA on 05 December 2017 as a GLP-1 receptor agonist and indicated as an adjunct to diet and exercise to improve glycemic control in adults with type 2 diabetes mellitus. OZEMPIC ® , 2 mg/1.5 ml.
- Semaglutide is chemically known as N- 26 -[2-(2-[2-(2-[2-(2-[2-(2-[4-(17-Carboxyhepta- decanoylamino)-4(S)-carboxybutyrylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl]
- Semaglutide can be represented by the following structural Formula I:
- WO2019120639A1 and W02020074583A2 reported the preparation of semaglutide via fermentation, solid phase peptide synthesis or fragmentation approaches.
- glucagon-like peptides are particularly demanding due to their propensity to aggregate. It is known that glucagon and glucagon-like peptides tend to aggregate at acidic pH (e.g. European J. Biochem. 11 (1969) 37-42).
- the present invention provides methods for the production and purification of semaglutide.
- Literature reported various purification methods like cation and anion-exchange purification process reported in US6451987B1, US6444788B1, ion-exchange chromatography in W02005019261A1, combination of ion-exchange and RP-HPLC by employing Tris as a buffering agent or an additive and organic modifiers in loading solution in US8710181, counter-current purification system in US9441028, RP-HPLC under involving pH adjustment in a step-wise manner in US9422330, using metal ions in US9447163, simulated chromatographic separations using mathematical model in US9766217.
- the present invention is related to a process for purification of semaglutide from a composition comprising semaglutide and one or more impurities, comprising the steps of: a) subjecting a composition comprising semaglutide and one or more impurities to first reversed phase HPLC purification, wherein a hydrocarbon bonded silica is used as a stationary phase, using mobile phase A, comprising aqueous basic phosphate buffer at a pH between about 7.5 to 8.5, and mobile phase B comprising organic solvent, and then eluting the desired peptide fractions; b) subjecting the pooled desired peptide fractions obtained in step a) to a second reversed phase HPLC purification, wherein a hydrocarbon bonded silica is used as a stationary phase, using mobile phase A, comprising acidic purification at a pH between about 2.5 to 3.5, and mobile phase B comprising organic solvent, and then eluting the desired peptide fractions; c) subjecting the pooled desired bonded silica
- the pure isolated semaglutide comprising phosphate content in the range between about 5 to about 15% w/w.
- the pure isolated semaglutide comprising phosphate content in the range between about 5 to about 15% w/w and sodium content in the range between about 4 to about 10% w/w.
- basic phosphate buffer can be selected from sodium dihydrogen phosphate, disodium hydrogen phosphate or sodium phosphate.
- the present invention is related to the pure isolated semaglutide comprising phosphate content in the range between about 5 to about 15% w/w.
- the present invention is related to the pure isolated semaglutide comprising phosphate content in the range between about 5 to about 15% w/w and sodium content in the range between about 4 to about 10% w/w.
- the present invention is related to stable semaglutide comprising phosphate content in the range between 5 to 15% w/w and sodium content in the range between about 4 to about 10% w/w.
- the present invention is related to the pure stable semaglutide comprising D-enantiomer content of amino acids-Aspartic acid, Phenylalanine, Glutamic acid, Tyrosine, Histidine in the range between less than about 0.05% to less than about 0.30% w/w, more preferably less than about 0.05% to less than about 0.20% w/w.
- the present invention is related to a process for purification of semaglutide from a composition comprising semaglutide and one or more impurities, comprising the steps of: a) subjecting a composition comprising semaglutide and one or more impurities to first reversed phase HPLC purification, wherein a hydrocarbon bonded silica is used as a stationary phase, using mobile phase A, comprising aqueous basic phosphate buffer at a pH between about 7.5 to 8.5, and mobile phase B comprising organic solvent, and then eluting the desired peptide fractions; b) subjecting the pooled desired peptide fractions obtained in step a) to a second reversed phase HPLC purification, wherein a hydrocarbon bonded silica is used as a stationary phase, using mobile phase A, comprising acidic purification at a pH between about 2.5 to 3.5, and mobile phase B comprising organic solvent, and then eluting the desired peptide fractions; c) subjecting the pooled desired bonded silica
- the mobile phase A comprising aqueous basic phosphate buffer at a pH between about 7.5 to 8.5.
- the suitable basic buffer can be selected but not limited from a group consisting of sodium dihydrogen phosphate, disodium hydrogen phosphate or sodium phosphate.
- the mobile phase B is organic solvent which can be selected from acetonitrile, C1-C4 alcohols or suitable mixture thereof.
- the step (b) is performed at acidic pH between about 2.5 to about 3.5.
- the acidic purification is performed in the presence of acidic buffer and suitable ion pairing agent.
- the suitable acidic buffer can be selected from ammonium formate and suitable ion pairing agent can be selected from TFA.
- the mobile phase B is organic solvent which can be selected from acetonitrile, C1-C4 alcohols, or suitable mixtures thereof.
- the pH of pooled desired peptide fractions as obtained in step (b) is adjusted to 8.0 ⁇ 0.5 by using suitable buffer.
- the suitable buffer can be selected but not limited from a group consisting offrom sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, potassium dihydrogen phosphate, ammonium bicarbonate or a combination thereof.
- the mobile phase A comprising aqueous basic phosphate buffer at a pH between about 7.5 to 8.5.
- the suitable basic buffer can be selected but not limited from a group consisting offrom sodium dihydrogen phosphate, disodium hydrogen phosphate or sodium phosphate.
- the mobile phase B is organic solvent which can be selected from acetonitrile, Cl- C4 alcohols or suitable mixture thereof.
- step (d) of the first embodiment involves isolation of pure semaglutide from the desired pooled fractions as obtained in step (c).
- the suitable isolation process for pure semaglutide can be selected from lyophilization or spray drying.
- pure semaglutide has purity greater than 99.0%.
- a crude liquid sample of semaglutide may be obtained from Solid Phase Peptide Synthesis (SPPS) or Liquid Phase Peptide Synthesis (LPPS) or a combination thereof.
- SPPS Solid Phase Peptide Synthesis
- LPPS Liquid Phase Peptide Synthesis
- the crude semaglutide was obtiained by Solid Phase Peptide Synthesis (SPPS) as reported in US8129343B2, CN104356224A with or without microwave technology.
- SPPS Solid Phase Peptide Synthesis
- the crude liquid sample of semaglutide obtained by above method can be isolated as dried peptide by well-known methods.
- a crude liquid sample of semaglutide is obtained by cleavage and deprotection of resin bound protected semglutide using DODT, TIPS, anisole in TFA and water.
- the crude sample of semaglutide thus obtained maybe extracted with organic solvent(s) such as Methyl tert-butyl ether (MTBE) and the like.
- organic solvent(s) such as Methyl tert-butyl ether (MTBE) and the like.
- the crude sample of semaglutide in buffer solution can be subjected to purification without drying and isolation as solid.
- the present invention relates to storage stable solution of semaglutide in buffer solution.
- the step (b) of the first embodiment involves purification of semaglutide from a composition comprising semaglutide and one or more impurities, wherein a composition comprising semaglutide and one or more impurities may be prepared from the crude dried semaglutide or the crude liquid sample of semaglutide.
- the step (b) of the first embodiment involves preparation of a composition comprising semaglutide and one or more impurities from crude dried semaglutide, comprising the step of:
- step (ii) heating the solution as obtained in step (i) to remove unwanted adduct
- step (iii) diluting the solution obtained in step (ii) with suitable solvent to give the composition comprising semaglutide and one or more impurities.
- the suitable buffer can be selected but not limited from a group consisting of from sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium phosphate, ammonium phosphate, ammonium carbonate, ammonium chloride, ammonium bicarbonate, ammonium sulphate, ammonium hydroxide, sodium acetate, sodium carbonate, sodium chloride, sodium bicarbonate, sodium phosphate and sodium sulphate, potassium carbonate, potassium acetate, or a combination thereof.
- the suitable solvent can be selected but not limited from a group consisting of from acetonitrile or C1-C4 alcohols, water or suitable mixtures thereof.
- the step (b) of the first embodiment involves preparation of a composition comprising semaglutide and one or more impurities from the crude liquid sample of semaglutide, comprising the step of:
- the suitable solvent can be selected but not limited from a group consisting of from acetonitrile, C1-C4 alcohols, water or suitable mixtures thereof.
- one or more impurities present in the composition comprising semaglutide are unwanted components which may formed during the synthesis of semaglutide.
- Particularly preferred types of impurities which formed during synthesis of semaglutide may exemplarily be selected from the group consisting of amino acids, peptides and derivatives thereof.
- impurities may also result from: premature chain termination during peptide synthesis, omission or unintended addition of at leastone amino acid during peptide synthesis, incomplete removal of protecting groups, side reactions occurring during amino acid coupling or Fmoc deprotection steps, inter or intramolecular condensation reactions, side reactions during peptide cleavage from a solid support, racemization, any other type of isomer formation, deamidation, (partial)hydrolysis, and aggregate formation.
- the pure isolated semaglutide comprising phosphate content in the range between about 5 to about 15% w/w.
- the pure isolated semaglutide comprising phosphate content in the range between about 5 to about 15% w/w and sodium content in the range between about 4 to about 10% w/w.
- the present invention is related to the pure isolated semaglutide comprising phosphate content in the range between about 5 to about 15% w/w.
- the present invention is related to the pure isolated semaglutide comprising phosphate content in the range between about 5 to about 15% w/w and sodium content in the range between about 4 to about 10% w/w.
- the present invention is related to the pure stable semaglutide comprising phosphate content in the range between about 5 to about 15% w/w and sodium content in the range between about 4 to about 10% w/w.
- the present invention is related to the pure stable semaglutide comprising D-enantiomer content of amino acids-Aspartic acid, Phenylalanine, Glutamic acid, Tyrosine, Histidine in the range between less than about 0.05% to less than about 0.30% w/w, more preferably less than about 0.05% to less than about 0.20% w/w.
- the pure isolated semaglutide is stable semaglutide which comprises phosphate content in the range between about 5 to about 15% w/w and sodium content in the range between about 4 to about 10% w/w.
- the stable semaglutide has improved physical stability at large scale specifically during holding or in-use period.
- the stable semaglutide has improved solubility of about 5.5 mg/ml in water.
- improved physical stability and solubility of stable semaglutide makes it more compatible for making injectable product.
- Nib refers to a-aminoisobutyric acid.
- TFA trifluoroacetic acid
- H3PO4 refers to phosphoric acid.
- purification is used to designate a process by which a composition comprising semaglutide and one or more impurities is purified.
- HPLC purity i.e.as relative peak area observed in analytical reversed phase high performance liquid chromatography (RP-HPLC) with UV detection at a wavelength between 205 and 230 nm, i.e. at the absorption maximum of the peptide bond.
- RP-HPLC reversed phase high performance liquid chromatography
- the value is determined as % area of a given peak area divided by the sum of the areas of all observed peaks in a chromatogram obtained by analytical RP-HPLC with UV detection at a wavelength between 205 and 230 nm.
- hydrocarbon bonded silica refers to stationary chromatographic phases made from porous silica particles or silica gels having chemically bonded hydrocarbon moieties at their surface. It is understood that the type of chemical bond as well as the chemical nature of the bonded hydrocarbon moieties may vary.
- a stationary phase for use with the present application may be made from porous silica particles having chemically bonded hydrocarbon moieties of 4 to 18, preferably 8 to 18, carbon atoms. Such hydrocarbon moieties are preferably linear alkyl chains.
- hydrocarbon bonded silica have hydrocarbon moieties with four (C4), six (C6), eight (C8), ten (CIO), twelve (C12), fourteen (C14), sixteen (C16), or eighteen (C18) carbon atoms.
- Particularly preferred types of hydrocarbon bonded silica have unbranched alkyl chains of four (C4), eight (C8), twelve (C12) or eighteen (Cl 8) carbon atoms, i.e. butyl, octyl, dodecyl, or octadecyl moieties.
- C8 bonded silica, in particular n-octyl bonded silica, and/or Cl 8 bonded silica, in particular n-octadecyl bonded silica are even more preferred stationary phases for use in steps a) and b) of a method according to the present invention.
- the stationary phase used in steps a) and b) may be the same or different in each of the steps.
- Preferably the stationary phase is the same.
- C8 bonded silica is used to designate stationary chromatographic phases made from porous silica particles or silica gels having at their surface chemically bonded C8 hydrocarbon moieties, preferably linear octyl, i.e. n-octyl, moieties.
- Cl 8 bonded silica or “ODS” are used herein interchangeably to refer to stationary chromatographic phases made from porous silica particles or silica gels having at their surface chemically bonded C18 hydrocarbon moieties, preferably linear octadecyl, i.e.
- n-octadecyl, moieties A wide range of hydrocarbon bonded silica materials is commercially available.
- stationary phases which can be used in present invention are DaisogelTM C18 ODS, Daiso ODS-Bio, Daiso-ODS-A-HG C18, DaisogelTM C8-Bio, YMC ODS-A, YMC Triart C8-L, Luna C8, Luna Cl 8, KromasilTM Cl 8, and KromasilTM C8 produced by Daiso, YMC, Phenomenex, and AkzoNobel, respectively.
- the silica particles may be of 2 to 200 micrometer, preferably 2.5 to 20micrometer, preferably 5- 15 micrometer, and most preferably 10 micrometer, indiameter and may have a pore size of 50 to 1000 A, preferably of 80 to 400 A, preferably of 100 to 300 A, most preferably of (about) lOOA.
- all or parts of the chromatographic purification are carried out at a temperature selected from the range of 10-30 °C, preferably 15-25°C.
- Example 1 Cleavage and extraction of crude semaglutide Resin bound protected semglutide (6 g) was charged into reactor containing DODT (2.52 mL), TIPS (0.36 mL), anisole (2.52 mL) in TFA (42 mL) and water (0.84 mL). The reaction mass was stirred for 5-20 minutes at 0-10 °C. The reaction mass temperature was raised to 10-20 °C and the reaction mass was maintained for 4-6 hours. The reaction mass was cooled to 0-10°C and water (120 mL) was charged into reaction mass. Ammonia solution (25% v/v, 54 mL) was charged and the reaction mass was quenched at 0-40°C.
- reaction mass was cooled to 25-35 °C.
- MTBE 60 x 3 mL was charged into the reaction mass and the reaction mass was stirred for 5-20 minutes at 25-35°C.
- the reaction mass was filtered and the filtrate was heated with stirring to 35-45°C for 1-2 hour.
- the layers were separated.
- Water 120 mL was charged into the aqueous layer and the reaction mass was cooled to 25-35°C.
- the pH of reaction mass was adjusted to pH 8.0 using 10% TFA solution (10% v/v, 4.5 mL) at 25-35°C to obtain crude semaglutide which was subjected to purification.
- the reverse phase media C18 bonded silica (10 micron) media was equilibrated with 90:10, 4m M disodium hydrogen phosphate pH 8.25 : acetonitrile.
- the feed containing crude semaglutide diluted with water is loaded onto the column.
- the crude peptide was then eluted from the column using a gradient elution, with the following mobile phases:
- Disodium Hydrogen Phosphate prepared by adding Disodium hydrogen phosphate, trifluoro acetic acid and sodium chloride into purified water), pH 8.25 (pH adjusted by liquid ammonia)
- the pure fractions obtained from second purification step were loaded onto the reverse phase Cl 8 (10 micron) column after equilibration with 90:10 4m M disodium hydrogen phosphate pH 8: acetonitrile.
- the feed is loaded onto the column and eluted from the column using a gradient elution, with the following mobile phases:
- Example 3 Purification of semaglutide a) Preparation of the composition comprising semaglutide and one or more impurities from dried peptide:
- the pure fractions obtained from first purification step were loaded onto the reverse phase C18 (10 micron) column after equilibration with 90:10 4mM disodium hydrogen phosphate pH 8: acetonitrile.
- the feed is loaded onto the column and washed with 90:10 4m M disodium hydrogen phosphate pH 8: acetonitrile for 30 min.
- the peptide was then eluted from the column using a gradient elution, with the following mobile phases:
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IN202041030673 | 2020-07-18 | ||
IN202141010313 | 2021-03-11 | ||
PCT/IN2021/050691 WO2022018748A1 (en) | 2020-07-18 | 2021-07-16 | Improved purification process of semaglutide |
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EP3000482B1 (de) * | 2009-12-16 | 2021-04-21 | Novo Nordisk A/S | Zweifach acylierte glp-1-derivate |
CN109456402A (zh) * | 2018-12-31 | 2019-03-12 | 江苏诺泰澳赛诺生物制药股份有限公司 | 一种索玛鲁肽的合成方法 |
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