CN114478750A - Purification method of teriparatide - Google Patents
Purification method of teriparatide Download PDFInfo
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- CN114478750A CN114478750A CN202111631316.7A CN202111631316A CN114478750A CN 114478750 A CN114478750 A CN 114478750A CN 202111631316 A CN202111631316 A CN 202111631316A CN 114478750 A CN114478750 A CN 114478750A
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- acetonitrile
- teriparatide
- purification method
- phosphate
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- 108010049264 Teriparatide Proteins 0.000 title claims abstract description 37
- 238000000746 purification Methods 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 29
- 229960005460 teriparatide Drugs 0.000 title claims abstract description 25
- OGBMKVWORPGQRR-UMXFMPSGSA-N teriparatide Chemical compound C([C@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)C(C)C)[C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=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](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CNC=N1 OGBMKVWORPGQRR-UMXFMPSGSA-N 0.000 title claims abstract description 25
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 105
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 51
- 238000010828 elution Methods 0.000 claims abstract description 32
- 150000003839 salts Chemical class 0.000 claims abstract description 19
- 239000000243 solution Substances 0.000 claims abstract description 18
- 238000005406 washing Methods 0.000 claims abstract description 14
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims abstract description 11
- 238000006243 chemical reaction Methods 0.000 claims abstract description 10
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 10
- 239000007853 buffer solution Substances 0.000 claims abstract description 9
- 229910019142 PO4 Inorganic materials 0.000 claims abstract description 8
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims abstract description 8
- 239000010452 phosphate Substances 0.000 claims abstract description 8
- 239000008055 phosphate buffer solution Substances 0.000 claims abstract description 8
- BUUKFBVDKSFMHN-LKMAISLMSA-N parathar acetate Chemical compound CC(O)=O.C([C@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)C(C)C)[C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=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](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CN=CN1 BUUKFBVDKSFMHN-LKMAISLMSA-N 0.000 claims abstract description 6
- 229960000338 teriparatide acetate Drugs 0.000 claims abstract description 6
- 238000004108 freeze drying Methods 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 13
- 238000011068 loading method Methods 0.000 claims description 8
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- 238000011282 treatment Methods 0.000 claims description 6
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 claims description 5
- 239000001099 ammonium carbonate Substances 0.000 claims description 5
- 238000012856 packing Methods 0.000 claims description 5
- 238000004237 preparative chromatography Methods 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
- 238000010790 dilution Methods 0.000 claims description 4
- 239000012895 dilution Substances 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
- 238000002390 rotary evaporation Methods 0.000 claims description 4
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 claims description 3
- 235000012538 ammonium bicarbonate Nutrition 0.000 claims description 3
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 claims description 3
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 claims description 3
- 239000000463 material Substances 0.000 claims description 3
- 235000019837 monoammonium phosphate Nutrition 0.000 claims description 3
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 3
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 3
- 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
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 2
- 229910000388 diammonium phosphate Inorganic materials 0.000 claims description 2
- 235000019838 diammonium phosphate Nutrition 0.000 claims description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 2
- 238000011067 equilibration Methods 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 2
- 239000011736 potassium bicarbonate Substances 0.000 claims description 2
- 229910000028 potassium bicarbonate Inorganic materials 0.000 claims description 2
- 235000015497 potassium bicarbonate 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
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 claims description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 2
- 235000017550 sodium carbonate Nutrition 0.000 claims description 2
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- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 208000001132 Osteoporosis Diseases 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 4
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 210000000963 osteoblast Anatomy 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 3
- 239000005695 Ammonium acetate Substances 0.000 description 3
- 108090000445 Parathyroid hormone Proteins 0.000 description 3
- 102000003982 Parathyroid hormone Human genes 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- 229940043376 ammonium acetate Drugs 0.000 description 3
- 235000019257 ammonium acetate Nutrition 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 239000007791 liquid phase Substances 0.000 description 3
- 239000000199 parathyroid hormone Substances 0.000 description 3
- 229960001319 parathyroid hormone Drugs 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 208000010392 Bone Fractures Diseases 0.000 description 2
- 206010017076 Fracture Diseases 0.000 description 2
- 108700032487 GAP-43-3 Proteins 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000005185 salting out Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 206010065687 Bone loss Diseases 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 208000007101 Muscle Cramp Diseases 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 201000001880 Sexual dysfunction Diseases 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-M Trifluoroacetate Chemical compound [O-]C(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-M 0.000 description 1
- PQLVXDKIJBQVDF-UHFFFAOYSA-N acetic acid;hydrate Chemical compound O.CC(O)=O PQLVXDKIJBQVDF-UHFFFAOYSA-N 0.000 description 1
- QQIJAZAJFULXMZ-UHFFFAOYSA-N acetonitrile;sulfuric acid Chemical compound CC#N.OS(O)(=O)=O QQIJAZAJFULXMZ-UHFFFAOYSA-N 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- KKTCWAXMXADOBB-UHFFFAOYSA-N azanium;hydrogen carbonate;hydrate Chemical compound [NH4+].O.OC([O-])=O KKTCWAXMXADOBB-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
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- OGBMKVWORPGQRR-UHFFFAOYSA-N forteo Chemical compound C=1NC=NC=1CC(C(=O)NC(CC(C)C)C(=O)NC(CC(N)=O)C(=O)NC(CO)C(=O)NC(CCSC)C(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NC(C(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC=1C2=CC=CC=C2NC=1)C(=O)NC(CC(C)C)C(=O)NC(CCCNC(N)=N)C(=O)NC(CCCCN)C(=O)NC(CCCCN)C(=O)NC(CC(C)C)C(=O)NC(CCC(N)=O)C(=O)NC(CC(O)=O)C(=O)NC(C(C)C)C(=O)NC(CC=1N=CNC=1)C(=O)NC(CC(N)=O)C(=O)NC(CC=1C=CC=CC=1)C(O)=O)NC(=O)C(CCCCN)NC(=O)CNC(=O)C(CC(C)C)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CCSC)NC(=O)C(CC(C)C)NC(=O)C(CCC(N)=O)NC(=O)C(NC(=O)C(CCC(O)=O)NC(=O)C(CO)NC(=O)C(NC(=O)C(N)CO)C(C)C)C(C)CC)CC1=CNC=N1 OGBMKVWORPGQRR-UHFFFAOYSA-N 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
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- 239000007790 solid phase Substances 0.000 description 1
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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
- C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Endocrinology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for purifying teriparatide. The method comprises the following steps: (1) taking phosphate buffer solution as a mobile phase A1, taking acetonitrile as a mobile phase B to carry out gradient elution on the crude peptide solution of teriparatide, and collecting elution components; (2) washing the elution component collected in the step (1) by taking carbonate buffer solution as a mobile phase A2 and acetonitrile as a mobile phase B to replace phosphate in the elution component; (3) salt conversion: and (3) performing gradient elution by taking an acetic acid solution as a mobile phase A3 and acetonitrile as a mobile phase B, and collecting the eluted components to obtain teriparatide acetate. According to the invention, a single impurity can be controlled within 0.10% through one-step purification, excessive organic solvent consumption caused by multiple purification is avoided, purification steps and waste liquid discharge are reduced, the whole process is simple to operate, the production period is short, the environment is friendly, and large-scale industrial production is facilitated.
Description
Technical Field
The invention relates to the technical field of polypeptide purification, in particular to a method for purifying teriparatide.
Background
Teriparatide (Teriparide), developed by American etiquette, is a 1-34 amino acid fragment (PTH1-34) of the biologically active N-terminal region of the 84 amino acid endogenous parathyroid hormone (PTH) of the formula C181H291N55O51S2Molecular weight 4177.77. The U.S. food and drug administration approved teriparatide for the treatment of osteoporosis with a high risk of fracture in men and women during menopause, as well as for the treatment of primary osteoporosis in men and or osteoporosis with a high risk of fracture due to sexual dysfunction on day 11, 26 of 2002. Teriparatide is the first new class of bone forming agents to obtain FDA approval from the U.S. food and drug administration. The conventional osteoporosis medicines only act on osteoclast to slow down or block bone loss, and teriparatide, a parathyroid hormone derivative, has the effects of increasing the number of osteoblast, enhancing the activity of the osteoblast and preventing the apoptosis of the osteoblast. It can increase activity and quantity of osteoblast, thus promoting bone growth, and can be used for second-line treatment of severe osteoporosis. In addition, teriparatide has few side effects, usually only nausea, dizziness and leg cramps. Therefore, the teriparatide has high medicinal value and wide market prospect. The peptide sequence is as follows:
H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH。
teriparatide synthesized by a solid phase has more racemic impurities, and because a peptide chain is longer, the separation difficulty of the racemic impurities from a main component is high, so that the impurities can be reduced to an acceptable level by multi-step purification in the purification process, the purification period is long, and the loss of a sample is large.
Such as: in CN 101798334B, obtaining teriparatide acetate by one-step purification and one-step salt transfer, using octadecylsilane chemically bonded silica as a stationary phase, using 0.05-0.2% trifluoroacetic acid aqueous solution as an A phase, using acetonitrile as a B phase, and performing gradient: b%: 20% -40%, and performing gradient elution on the crude peptide solution; then, the trifluoroacetate of human parathyroid hormone (1-34) is converted into acetate by reverse phase high performance liquid chromatography. The purity of teriparatide obtained by the method is 98%, and the racemization impurities are not controlled.
In CN 102993293A, a solution obtained by adjusting the pH value of 0.1-0.4% sulfuric acid and 0.1-0.4% acetic acid aqueous solution by volume ratio with ammonia water to 5.0-6.0 is used as an A phase, and acetonitrile is used as a B phase for purification; then salting out the qualified fraction; washing with ammonium acetate solution containing 3-10% acetonitrile, transferring salt with octadecyl silane bonded silica gel high performance liquid chromatography, and eluting with acetic acid water solution acetonitrile system. After salting out, 50% -100% phosphoric acid aqueous solution is used for dissolving the salt, so that the high-concentration phosphoric acid cannot be subjected to next salt conversion, alkali is used for neutralization, more degradation impurities are generated in the neutralization process, and the dissolution process has large loss.
In CN 106167522 a, ammonium acetate was used for purification and then converted to acetate, the process was only controlled to a purity of > 99.0%, without control of racemic impurities.
CN 108373499A, the crude peptide solution is purified for the first time by using an acetonitrile system of ammonium bicarbonate water solution; secondly, purifying the collected eluent for the second time by using a sodium sulfate/sulfuric acid acetonitrile system; thirdly, eluting the collected eluent by using an ammonium acetate aqueous solution acetonitrile system, and then eluting by using an acetic acid aqueous solution acetonitrile system to carry out high-efficiency liquid phase salt conversion; and fourthly, decompressing and concentrating the eluent after salt conversion, and then adding hydrochloric acid. The method adopts multiple purification steps and different chromatographic systems, so that the steps are complicated, the consumption of the purified acetonitrile is high, and the method is not favorable for environmental protection.
Disclosure of Invention
Aiming at the technical problems of unsatisfactory purification effect of teriparatide, poor control of racemic impurities, complicated purification steps and the like in the background technology, the invention provides a purification method of teriparatide, and an acetate sample with the purity of more than or equal to 99.0 percent and the single impurity of less than or equal to 0.10 percent is obtained through one-step purification. And the single sample loading is high, the production period is short, the waste liquid amount is small, and the environment is protected.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides a method for purifying teriparatide, which comprises the following steps:
(1) taking phosphate buffer solution as a mobile phase A1, taking acetonitrile as a mobile phase B to carry out gradient elution on the crude peptide solution of teriparatide, and collecting elution components;
(2) washing and displacing phosphate in the elution component collected in the step (1) by using carbonate buffer solution as a mobile phase A2 and acetonitrile as a mobile phase B;
(3) salt conversion: and (3) performing gradient elution by taking an acetic acid solution as a mobile phase A3 and acetonitrile as a mobile phase B, and collecting the eluted components to obtain teriparatide acetate.
In a preferable embodiment, in the step (1), the initial volume percentage of the acetonitrile in the mobile phase B is 15-25%, the final volume percentage is 30-40%, and the elution time is 60-100 min.
Preferably, in the step (1), the phosphate is selected from any one or any mixture of ammonium dihydrogen phosphate, diammonium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate.
Preferably, in the step (1), the concentration of the phosphate buffer solution is 5-500 mmol/L in terms of the concentration of phosphate; more preferably 20 to 200 mmol/L.
In certain embodiments, the phosphate buffer has a concentration of 5mmol/L, 50mmol/L, 100mmol/L, 200mmol/L, 300mmol/L, 400mmol/L, 500mmol/L or any value therebetween, based on the concentration of phosphate.
Preferably, in the step (1), the pH of the phosphate buffer solution is 6.0-8.0.
In certain specific embodiments, the pH of the phosphate buffer is 6.0, 6.5, 7.0, 7.2, 7.5, 7.7, 8.0, or any value therebetween.
In certain embodiments, the phosphate buffer is formulated by mixing phosphoric acid with ammonia, sodium hydroxide, or potassium hydroxide.
As a preferred embodiment, in the step (1), the crude teriparatide peptide solution is a mixed solution of crude teriparatide peptide dissolved in acetonitrile and water; the volume percentage of the acetonitrile is 5-15%.
In a preferable embodiment, in the step (2), the volume percentage of the acetonitrile in the mobile phase B is 5-15%, and the washing time is 10-30 min;
as a preferred embodiment, in the step (2), the carbonate is any one or more selected from ammonium carbonate, ammonium bicarbonate, sodium carbonate, sodium bicarbonate, potassium carbonate and potassium bicarbonate.
Preferably, in the step (2), the concentration of the carbonate buffer solution is 5-500 mmol/L; more preferably 20 to 200 mmol/L.
In certain embodiments, the carbonate buffer solution has a concentration of 5mmol/L, 50mmol/L, 100mmol/L, 200mmol/L, 300mmol/L, 400mmol/L, 500mmol/L, or any value therebetween.
Preferably, in the step (2), the carbonate buffer solution is adjusted to have a pH of 6.0-8.0 by acetic acid.
In certain embodiments, the carbonate buffer solution is adjusted to a pH of 6.0, 6.5, 7.0, 7.2, 7.5, 7.7, 8.0, or any value therebetween with acetic acid.
In some specific embodiments, in the step (2), before the washing, the elution component obtained in the step (1) needs to be diluted; the dilution is carried out by using water, and specifically is carried out by using 0.8-1.5 times of water by volume.
In a preferable embodiment, in the step (3), the initial volume percentage of the acetonitrile in the mobile phase B is 5-15%, the final volume percentage is 50-70%, and the elution time is 30-60 min.
In a preferred embodiment, in the step (3), the concentration of the acetic acid solution is 0.005% to 0.05%.
In certain specific embodiments, the concentration of the acetic acid solution is 0.005%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, or any number therebetween.
In certain specific embodiments, in step (3), the gradient elution further comprises a pretreatment, wherein the pretreatment is washing with the mobile phase a3 and the mobile phase B.
Preferably, the washing time is 10-30 min.
In the technical scheme of the invention, the purification method adopts a reversed-phase preparation chromatographic column; the inner packing of the reversed-phase preparative chromatographic column is selected from any one of reversed-phase C18, C8 and C4;
preferably, the particle size of the internal filler is 5-10 μm; the aperture of the internal filler is 5-30 nm.
In a preferred embodiment, in the step (1), the step (2) and the step (3), the loading amount of the reverse phase preparative chromatography column is 0.5 to 5.0% by mass of the packing material contained therein, and more preferably 1.0 to 3.0%.
In certain specific embodiments, in step (1), the gradient elution further comprises a pretreatment comprising filtration and equilibration of the preparative chromatography column; the equilibrium preparative chromatographic column is a chromatographic column prepared by balancing the mobile phase A1 and the mobile phase B, wherein the volume percentage of the mobile phase B is 5-15%, the volume percentage of acetonitrile in the preparative chromatographic column is smaller than the initial elution gradient by adopting a proper mobile phase proportion, and a sample to be processed can be just adsorbed on the chromatographic column without being eluted.
In certain specific embodiments, step (3) further comprises post-treatment, wherein the post-treatment comprises reduced pressure rotary evaporation and freeze drying.
The technical scheme has the following advantages or beneficial effects: according to the purification method of teriparatide, phosphate buffer solution is used as a mobile phase A1, acetonitrile is used as a mobile phase B to carry out gradient elution on a crude teriparatide peptide solution, a qualified sample with the purity of more than or equal to 99.0% and the single impurity of less than or equal to 0.10% can be obtained through one-step purification, and then the qualified sample is converted into acetate. According to the invention, a single impurity can be controlled within 0.10% through one-step purification, excessive organic solvent consumption caused by multiple purification is avoided, purification steps and waste liquid discharge are reduced, the whole process is simple to operate, the production period is short, the environment is friendly, and large-scale industrial production is facilitated.
Drawings
FIG. 1 is an RP-HPLC chromatogram of the protamine after the salt-transfer lyophilization in example 1.
FIG. 2 is an RP-HPLC chromatogram of the protamine after the salt-transfer lyophilization in example 2.
FIG. 3 is an RP-HPLC chromatogram of the protamine after transsalt lyophilization in example 3.
FIG. 4 is an RP-HPLC chromatogram of the protamine after salt transfer lyophilization in example 4.
FIG. 5 is an RP-HPLC chromatogram of the protamine after transsalt lyophilization in example 5.
FIG. 6 is an RP-HPLC chromatogram of the protamine after salt transfer lyophilization in comparative example 1.
FIG. 7 is an RP-HPLC chromatogram of the protamine after the lyophilisation of the salt in comparative example 2.
FIG. 8 is an RP-HPLC chromatogram of the protamine after salt transfer lyophilization in comparative example 3.
Detailed Description
The following examples are only a part of the present invention, and not all of them. Thus, the detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the invention without making creative efforts, belong to the protection scope of the invention.
In the present invention, all the equipment, materials and the like are commercially available or commonly used in the industry, if not specified. The methods in the following examples are conventional in the art unless otherwise specified.
In the following examples and comparative examples, crude teriparatide was prepared by gene recombination, solid phase synthesis, liquid phase synthesis or solid-liquid phase combination synthesis.
Example 1:
120.0g of crude teriparatide (containing 49.5g of teriparatide) is weighed out and dissolved in 3L of acetonitrile/water with a volume percentage of 10%, and filtered through a 0.45 μm filter membrane. The preparation column was 15cm DAC filled with 3.0kg of C18 filler (C)
10 μm is the particle diameter,
The same applies to the pore diameter). Mobile phase a1 was 50mmol/L ammonium dihydrogen phosphate in water, pH adjusted to 7.5 with ammonia, and mobile phase B was acetonitrile. Balancing 90% A1+ 10% B for 10min, flowing at 500mL/min, loading, gradient eluting with 25% B-35% B/(100min), collecting fractions with purity not less than 99.0% and single impurity not more than 0.10%, mixing the fractions with purity not less than 70.0% and not more than 99.0% or purity not less than 99.0% and single impurity more than 0.10%, loading, gradient eluting with A1 and B, and purifying.
And collecting all qualified fractions, adding water with the same volume for dilution, and performing salt conversion. Mobile phase a2 was 80mmol/L aqueous ammonium bicarbonate adjusted to pH 7.0 with acetic acid, mobile phase B was acetonitrile, and mobile phase A3 was 0.01% aqueous acetic acid. Washing with 90% A2+ 10% B for 10min after sample loading, with flow rate of 500 mL/min; switching A2 to A3 (0.01% acetic acid aqueous solution), washing for 10min with 90% A3+ 10% B at a flow rate of 500mL/min, then carrying out gradient elution with A3 and B, wherein the elution gradient is 10% B-60% B/(50min), collecting fractions with the purity of more than or equal to 99.0% and the single impurity of less than or equal to 0.10%, carrying out freeze-drying after rotary evaporation to obtain 32.0g of teriparatide acetate, wherein the purification recovery rate is 64.6%, the purity is 99.92%, and the maximum single impurity is 0.02%.
FIG. 1 is a test pattern of the fine peptide after the salt conversion in this example.
Example 2:
122.0g of crude teriparatide (containing 50.3g of teriparatide) is weighed out and dissolved in 3L acetonitrile/water with a volume percentage of 10%, and filtered through a 0.45 μm filter membrane. The preparation column was a 15cm DAC filled with 3.0kg of C8 packing
Mobile phase a1 was 50mmol/L sodium dihydrogen phosphate in water, pH adjusted to 6.0 with 50mmol/L sodium hydroxide in water, and mobile phase B was acetonitrile. Balancing 90% A1+ 10% B for 10min at flow rate of 500mL/min, performing gradient elution after sample loading,the elution gradient is 25 percent B-35 percent B/(100min), fractions with the purity of more than or equal to 99.0 percent and the single impurity of less than or equal to 0.10 percent are collected, the fractions with the purity of more than or equal to 70.0 percent and less than or equal to 99.0 percent or the fractions with the purity of more than or equal to 99.0 percent and the single impurity of more than 0.10 percent are combined, and after sampling, A1 and B are used for gradient elution and purification.
And collecting all qualified fractions, adding water with the same volume for dilution, and performing salt conversion. Mobile phase a2 was 80mmol/L aqueous sodium bicarbonate adjusted to pH 7.0 with acetic acid, mobile phase B was acetonitrile, and mobile phase A3 was 0.02% aqueous acetic acid. Washing with 90% A2+ 10% B for 10min after sample loading, with flow rate of 500 mL/min; switching A2 to be A3 (0.01% acetic acid aqueous solution), washing for 10min with 90% A3+ 10% B at a flow rate of 500mL/min, then carrying out gradient elution with A3 and B, wherein the elution gradient is 10% B-60% B/(50min), collecting fractions with the purity of more than or equal to 99.0% and the single impurity of less than or equal to 0.10%, carrying out rotary evaporation and freeze-drying to obtain 32.3g of teriparatide acetate, wherein the purification recovery rate is 64.2%, the purity is 99.95%, and the maximum single impurity is 0.04%.
FIG. 2 is a test pattern of the fine peptide after the salt conversion in this example.
Examples 3 to 5 and comparative examples 1 to 3
The Applicant has carried out the following examples and comparative examples simultaneously, preparing columns each of 15cm DAC, containing 3.0kg of C18 filler
The differences from the above examples 1 and 2 are summarized in Table 1.
TABLE 1
In examples 3-5, the test patterns of the protamine after the salt transfer and freeze drying are respectively shown in figures 3-5; the test patterns of the protamine after the salt transfer lyophilization in comparative examples 1 to 3 are shown in FIGS. 6 to 8, respectively.
The above description is only for the preferred embodiment of the present invention and is not intended to limit the scope of the present invention, and all equivalent modifications made by the contents of the present specification and the drawings, or applied directly or indirectly to other related technical fields, are included in the scope of the present invention.
Claims (10)
1. A method for purifying teriparatide, which is characterized by comprising the following steps:
(1) taking phosphate buffer solution as a mobile phase A1, taking acetonitrile as a mobile phase B to carry out gradient elution on the crude peptide solution of teriparatide, and collecting elution components;
(2) washing and displacing phosphate in the elution component collected in the step (1) by using carbonate buffer solution as a mobile phase A2 and acetonitrile as a mobile phase B;
(3) salt conversion: and (3) performing gradient elution by taking an acetic acid solution as a mobile phase A3 and acetonitrile as a mobile phase B, and collecting the eluted components to obtain teriparatide acetate.
2. The purification method according to claim 1, wherein in the step (1), the initial volume percentage of the acetonitrile in the mobile phase B is 15-25%, the initial volume percentage is 30-40%, and the elution time is 60-100 min;
preferably, in the step (1), the phosphate is selected from any one or any mixture of ammonium dihydrogen phosphate, diammonium hydrogen phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate and dipotassium hydrogen phosphate;
preferably, in the step (1), the concentration of the phosphate buffer solution is 5-500 mmol/L in terms of the concentration of phosphate; further preferably 20 to 200 mmol/L;
preferably, in the step (1), the pH of the phosphate buffer solution is 6.0-8.0.
3. The purification method according to claim 1, wherein in the step (1), the crude teriparatide peptide solution is a mixed solution of crude teriparatide peptide dissolved in acetonitrile and water; the volume percentage of the acetonitrile is 5-15%.
4. The purification method according to claim 1, wherein in the step (2), the volume percentage of the acetonitrile in the mobile phase B is 5-15%, and the washing time is 10-30 min.
5. The purification method according to claim 1, wherein in the step (2), the carbonate is selected from any one or more of ammonium carbonate, ammonium bicarbonate, sodium carbonate, sodium bicarbonate, potassium carbonate and potassium bicarbonate;
preferably, in the step (2), the concentration of the carbonate buffer solution is 5-500 mmol/L; further preferably 20 to 200 mmol/L;
preferably, in the step (2), the pH value of the carbonate buffer solution is adjusted to 6.0-8.0 by acetic acid;
preferably, in the step (2), before the washing, the elution component obtained in the step (1) needs to be diluted; the dilution is carried out by using water, and specifically is carried out by using 0.8-1.5 times of water by volume.
6. The purification method according to claim 1, wherein in the step (3), the initial volume percentage of the acetonitrile in the mobile phase B is 5-15%, the initial volume percentage is 50-70%, and the elution time is 30-60 min;
preferably, in the step (3), the concentration of the acetic acid solution is 0.005% -0.05%.
7. The purification method according to claim 1, wherein the purification method employs a reverse phase preparative chromatography column; the inner packing of the reversed-phase preparative chromatographic column is selected from any one of reversed-phase C18, C8 and C4;
preferably, the particle size of the internal filler is 5-10 μm; the aperture of the internal filler is 5-30 nm.
8. The purification method according to claim 7, wherein the loading amount of the reverse phase preparative chromatography column in the step (1), the step (2) and the step (3) is 0.5 to 5.0%, more preferably 1.0 to 3.0% of the mass of the packing material therein.
9. The purification method according to claim 1, wherein in step (1), the gradient elution further comprises a pretreatment comprising filtration and equilibration of the preparative chromatography column; the equilibrium preparative chromatographic column is an equilibrium preparative chromatographic column with the mobile phase A1 and the mobile phase B, wherein the volume percentage of the mobile phase B is 5-15%.
10. The purification method according to claim 1, wherein the step (3) further comprises post-treatment, and the post-treatment comprises reduced pressure rotary evaporation and freeze drying.
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