CN108373499A - A kind of purifying of Teriparatide acetate and ionic control method - Google Patents
A kind of purifying of Teriparatide acetate and ionic control method Download PDFInfo
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- CN108373499A CN108373499A CN201810119038.9A CN201810119038A CN108373499A CN 108373499 A CN108373499 A CN 108373499A CN 201810119038 A CN201810119038 A CN 201810119038A CN 108373499 A CN108373499 A CN 108373499A
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- teriparatide
- acetate
- purifying
- acetonitrile
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- 108010049264 Teriparatide Proteins 0.000 title claims abstract description 68
- 238000000034 method Methods 0.000 title claims abstract description 39
- 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 title claims abstract description 25
- 229960000338 teriparatide acetate Drugs 0.000 title claims abstract description 25
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 129
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 66
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 56
- 239000000243 solution Substances 0.000 claims abstract description 55
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims abstract description 47
- 229960005460 teriparatide Drugs 0.000 claims abstract description 43
- 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 claims abstract description 43
- 150000003839 salts Chemical class 0.000 claims abstract description 38
- 238000010828 elution Methods 0.000 claims abstract description 23
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims abstract description 22
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims abstract description 22
- 239000012535 impurity Substances 0.000 claims abstract description 19
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000007864 aqueous solution Substances 0.000 claims abstract description 17
- QEFRNWWLZKMPFJ-ZXPFJRLXSA-N L-methionine (R)-S-oxide Chemical compound C[S@@](=O)CC[C@H]([NH3+])C([O-])=O QEFRNWWLZKMPFJ-ZXPFJRLXSA-N 0.000 claims abstract description 14
- QEFRNWWLZKMPFJ-UHFFFAOYSA-N L-methionine sulphoxide Natural products CS(=O)CCC(N)C(O)=O QEFRNWWLZKMPFJ-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000003480 eluent Substances 0.000 claims abstract description 14
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 claims abstract description 12
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 claims abstract description 12
- 235000012538 ammonium bicarbonate Nutrition 0.000 claims abstract description 12
- 239000001099 ammonium carbonate Substances 0.000 claims abstract description 12
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000005695 Ammonium acetate Substances 0.000 claims abstract description 10
- 229940043376 ammonium acetate Drugs 0.000 claims abstract description 10
- 235000019257 ammonium acetate Nutrition 0.000 claims abstract description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims abstract description 9
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims abstract description 8
- RQBBFKINEJYDOB-UHFFFAOYSA-N acetic acid;acetonitrile Chemical compound CC#N.CC(O)=O RQBBFKINEJYDOB-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000007791 liquid phase Substances 0.000 claims abstract description 4
- 229910052938 sodium sulfate Inorganic materials 0.000 claims abstract description 4
- 235000011152 sodium sulphate Nutrition 0.000 claims abstract description 4
- 239000012071 phase Substances 0.000 claims description 85
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 46
- 239000000377 silicon dioxide Substances 0.000 claims description 23
- 230000005526 G1 to G0 transition Effects 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical group CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 16
- 150000002500 ions Chemical class 0.000 claims description 10
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 claims description 9
- 150000001343 alkyl silanes Chemical group 0.000 claims description 9
- 239000000908 ammonium hydroxide Substances 0.000 claims description 9
- LCPVQAHEFVXVKT-UHFFFAOYSA-N 2-(2,4-difluorophenoxy)pyridin-3-amine Chemical compound NC1=CC=CN=C1OC1=CC=C(F)C=C1F LCPVQAHEFVXVKT-UHFFFAOYSA-N 0.000 claims description 8
- CHQMHPLRPQMAMX-UHFFFAOYSA-L sodium persulfate Substances [Na+].[Na+].[O-]S(=O)(=O)OOS([O-])(=O)=O CHQMHPLRPQMAMX-UHFFFAOYSA-L 0.000 claims description 8
- 239000000706 filtrate Substances 0.000 claims description 7
- 239000000499 gel Substances 0.000 claims description 7
- 238000005374 membrane filtration Methods 0.000 claims description 7
- 229910001220 stainless steel Inorganic materials 0.000 claims description 7
- 239000010935 stainless steel Substances 0.000 claims description 7
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims 1
- 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 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 claims 1
- 229910052739 hydrogen Inorganic materials 0.000 claims 1
- 239000001257 hydrogen Substances 0.000 claims 1
- 125000004435 hydrogen atom Chemical class [H]* 0.000 claims 1
- 229910052708 sodium Inorganic materials 0.000 claims 1
- 239000011734 sodium Substances 0.000 claims 1
- 238000011084 recovery Methods 0.000 abstract description 23
- 239000000523 sample Substances 0.000 description 43
- 238000000746 purification Methods 0.000 description 25
- 229960000583 acetic acid Drugs 0.000 description 21
- 238000004108 freeze drying Methods 0.000 description 17
- 238000011049 filling Methods 0.000 description 15
- 238000011068 loading method Methods 0.000 description 15
- 239000012488 sample solution Substances 0.000 description 15
- 239000000126 substance Substances 0.000 description 15
- 238000001514 detection method Methods 0.000 description 12
- 238000004587 chromatography analysis Methods 0.000 description 11
- 239000000047 product Substances 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 5
- 238000010829 isocratic elution Methods 0.000 description 5
- 230000014759 maintenance of location Effects 0.000 description 5
- 150000002825 nitriles Chemical class 0.000 description 5
- 238000002604 ultrasonography Methods 0.000 description 5
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000003292 glue Substances 0.000 description 3
- -1 salt Ion Chemical class 0.000 description 3
- 241000370738 Chlorion Species 0.000 description 2
- 108090000445 Parathyroid hormone Proteins 0.000 description 2
- 102000003982 Parathyroid hormone Human genes 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 2
- 150000001450 anions Chemical class 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000199 parathyroid hormone Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 2
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 101001135770 Homo sapiens Parathyroid hormone Proteins 0.000 description 1
- 101001135995 Homo sapiens Probable peptidyl-tRNA hydrolase Proteins 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- YDCNECXDBVLHLS-UHFFFAOYSA-N acetonitrile;azane Chemical compound N.CC#N YDCNECXDBVLHLS-UHFFFAOYSA-N 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- XORXDJBDZJBCOC-UHFFFAOYSA-N azanium;acetonitrile;acetate Chemical compound [NH4+].CC#N.CC([O-])=O XORXDJBDZJBCOC-UHFFFAOYSA-N 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 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
- 102000058004 human PTH Human genes 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 229960001319 parathyroid hormone Drugs 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 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
- C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
Landscapes
- 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 present invention provides purifying and the ionic control method of a kind of Teriparatide acetate, includes the following steps：1)：Thick peptide solution is subjected to first time purifying using the acetonitrile system of ammonium bicarbonate aqueous solution；2)：The eluent of above-mentioned collection is carried out second using the acetonitrile system of sodium sulphate/sulfuric acid to purify；3)：First ammonium acetate aqueous solution acetonitrile system is used to elute the eluent of collection, then carries out efficient liquid phase with the elution of aqueous acetic acid acetonitrile system and turn salt；4)：To turn that hydrochloric acid is added after the eluent after salt is concentrated under reduced pressure.The present invention has the following advantages：It is combined by addition hydrochloric acid progress acetate plasma content control after repeatedly purifying and turning salt; use different chromatographic systems; the Teriparatide acetic acid purity salt that prepare with scale obtains is more than 99%; methionine sulfoxide Teriparatide impurity summation≤0.50% and maximum unknown list it is miscellaneous≤0.10%; acetate ion content≤5%; ≤ 0.1%, total recovery is more than 60% for chloride ion content≤4%, trifluoroacetic acid root and sulfate radical content.
Description
Technical field
The present invention relates to a kind of purification process of polypeptide, more particularly to meet the Teriparatide acetic acid of 40 quality requirements of USP
The purifying of salt and ionic control method.
Background technology
Teriparatide (PTH (1-34)), English name Teriparatide is a kind of polypeptide drugs of long-chain, and peptide sequence is：
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 is the 1-34 amino acid fragments of human parathyroid hormone (PTH), by Eli Lilly companies of the U.S. through gene
Recombinant technique research and development are made, and drug is made in the form of acetate, listing was approved by the FDA in the United States in 2002 years.It is first
It is approved for the bone formation-promoter for the treatment of severe osteoporosis, has a vast market foreground.
Document and the patent for being related to Teriparatide synthesis at present are more, are related to isolating and purifying less, especially this hair
Bright purifies for the first time using the progress of high pressure reverse-phase chromatography and purifies for the second time and hydrochloric acid progress acetate etc. is added after turning salt
Ion concentration control is combined prepare with scale (a batch can obtain 200 grams of finished product or more) purity and ion concentration meets USP 40
Middle Teriparatide acetic acid salt bulk drug quality requirement be even more without reference to.
For the purification process of this product, existing patented technology carries out disclosure.Chinese patent CN106167522A disclose by
Crude product is dissolved in 30% acetonitrile solution, is purified with ammonium acetate buffer, and control pH is 4.0~7.0, acetic acid/acetonitrile C18 columns
Turn salt, the method that teriparatide acetate is made, although the teriparatide acetate acetate that purity reaches 99% or more can be obtained,
Single miscellaneous content is not pointed out, while acetic acid radical content is not described in final teriparatide acetate obtained.
Chinese patent CN102993293A disclose by sulfuric acid and acetic acid mixed aqueous solution ammonium hydroxide tune pH value to 5.0~
6.0 buffer solution is purified, and the sample for collecting 95%~99% adjust pH value to cool down to putting after 5.5~7.0 to 2~8 DEG C
Product is precipitated, is collected by centrifugation after precipitation with after 60% phosphate aqueous solution, then be diluted with water one times, turns salt after upper reversed-phase column, finally
Teriparatide acetate is made.Its yield does not embody in the patent.Sample is dissolved with 60% phosphoric acid before turning salt, and it is unstable that there are products
It degrades, introduce the unstable risk of new impurity, yield.Acetic acid radical content is not retouched in final teriparatide acetate obtained
It states, the content of other anion used in technique is not also studied.
Patent CN102731643A discloses a kind of purification process of Teriparatide, using the thick peptide of water dissolution, then uses
0.2%TFA/ acetonitriles are mobile phase, C18 column separating purifications, and 0.2% acetic acid/acetonitrile C18 columns turn salt, total recovery 20.5~
32.2%, do not study the concrete content of anion in maximum single miscellaneous and teriparatide acetate equally yet.
To solve impurity of the existing technology and the indefinite problem of ion concentration, acquisition meets 40 quality requirements of USP
Teriparatide bulk pharmaceutical chemicals, also need to conduct further research to purifying and turning salt method.
Invention content
The purpose of the present invention is to provide a kind of extensive works isolated and purified with ionic control of Teriparatide acetate
Process, purity is high and yield is good, content≤5% of acetate after freeze-drying, obtains the Te Lipa for meeting 40 quality requirements of USP
Peptide bulk pharmaceutical chemicals reach the requirement of industrialized production, solve the indefinite difficulty of impurity and ion concentration existing in the prior art
Topic
To achieve the goals above, the present invention provides the following technical solution：A kind of purifying of Teriparatide acetate and
Ionic control method, includes the following steps：
Step 1)：Thick peptide solution is subjected to first time gradient elution purifying using chromatographic column, collects eluent；The chromatography
The mobile phase that column uses includes mobile phase A and Mobile phase B, and the mobile phase A is ammonium bicarbonate aqueous solution, with acetic acid or ammonium hydroxide tune
PH, the Mobile phase B are acetonitrile；
Step 2)：The eluent of above-mentioned collection is subjected to second of gradient elution purifying using chromatographic column, collects eluent；
The mobile phase that the chromatographic column uses includes mobile phase A and Mobile phase B, and the mobile phase A is aqueous sodium persulfate solution, with sulfuric acid tune
PH, the Mobile phase B are acetonitrile；
Step 3)：The eluent collected in step 2) is subjected to efficient liquid phase using chromatographic column and turns salt, wherein first using acetic acid
Aqueous ammonium acetonitrile system elutes, then is eluted with aqueous acetic acid acetonitrile system；
Step 4)：Hydrochloric acid is added after eluent after step 3) transfer salt is concentrated under reduced pressure, makes the residual quantity control of acetate
System is below 5%.
Preferably, in the step 1) mobile phase A pH be 7.0~9.5, ammonium hydrogen carbonate a concentration of 0.01~
0.2mol/L。
As further preferred, the pH of mobile phase A is 8.0 in the step 1), a concentration of 0.1mol/ of ammonium hydrogen carbonate
L。
Preferably, in the step 2) mobile phase A pH be 2.0~4.0, sodium sulphate a concentration of 0.05~
0.2mol/L。
As further preferred, the pH of mobile phase A is 3.0 in the step 2), a concentration of 0.1mol/L of sodium sulphate.
Preferably, the amount for adjusting hydrochloric acid used in acetic acid radical content in the step 4) is 1~4N.
As further preferred, the amount that hydrochloric acid used in acetic acid radical content is adjusted in the step 4) is 3.3N.
As further preferred, the target peptide solution that 1~4 equivalent 1N hydrochloric acid is added is transferred to size in the step 4)
It is freeze-dried in suitable stainless steel pallet, obtained Teriparatide purity is more than 99%, methionine sulfoxide Teriparatide
Impurity summation≤0.50% and maximum unknown list it is miscellaneous≤0.10% teriparatide acetate, acetate ion content≤5%, chlorine from
≤ 0.1%, purifying yield is 60% for sub- content≤4%, trifluoroacetic acid root and sulfate radical content.
Preferably, the stationary phase that the step 1) to chromatographic column described in step 3) uses is octadecylsilane bonded silica
Glue or eight alkyl silane bonded silica gels.
Preferably, further comprising the steps：In above-mentioned steps 1) following sample treatment is carried out before, by thick peptide
It is dissolved according to 5~20g/L of concentration using the acetonitrile solution of volume ratio 5~10%, then uses 0.22~0.45 μm of filter membrane
It is for use to collect filtrate for filtering.
As a kind of further preferred, extensive side isolated and purified with ionic control of Teriparatide acetate
Method specifically includes following steps：
Step 1)：Thick peptide is dissolved according to concentration 5~20g/L with the acetonitrile solution of volume ratio 5~10%, using 0.22~
0.45 μm of membrane filtration.
Step 2)：First separation purifying is carried out to filtered crude product using high performance liquid chromatograph device：Pass through DAC-
CXTH200 prepares dynamic axial pressured column, by thick peptide solution with octadecylsilane chemically bonded silica or eight alkyl silane bonded silicas
Glue is that the chromatographic column progress of stationary phase is gradient-purified, by 0.01~0.2mol/L ammonium bicarbonate aqueous solutions ammonium hydroxide tune pH value 7.0
Solution after~9.5 is A phases, and acetonitrile is B phases, gradient：B%：10%~30% elution；Flow velocity is 1000~1400ml/min,
Detection wavelength is 220nm, Fractional Collections.
Step 3)：Dynamic axial pressured column is prepared by acetonitrile in the qualified solution collected in step 2 by DAC-CXTH200
Ratio be down to 10% and carry out second directly below and purify, with octadecylsilane chemically bonded silica or eight alkyl silane bonded silicas
Glue is that the chromatographic column of stationary phase carries out gradient elution purifying, is used with the aqueous sodium persulfate solution that molar concentration is 0.05~0.2mol/L
Solution after sulfuric acid tune pH value 2.0~4.0 is A phases, and acetonitrile is B phases, gradient：B%：10~30% elutions；Flow velocity be 1000~
1400ml/min, Detection wavelength 220nm, Fractional Collections.
Step 4)：Efficient liquid phase, which is carried out, with octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels turns salt, it will
Step 3) acquired solution rinses 20~30min, a concentration of 10~100mM of ammonium acetate with 3~5% acetonitrile ammonium acetate solution,
Then it is eluted with aqueous acetic acid acetonitrile system, collection teriparatide solutions, the volume ratio of acetic acid is in the aqueous acetic acid
0.1~0.2%, acetonitrile ratio is 35% or more.
Step 5)：It will turn 35 DEG C of eluent after salt or less to be concentrated under reduced pressure, the hydrochloric acid that 1~4N is added adjusts product after freeze-drying
Middle acetate ion content makes the residual of acetate control 5% hereinafter, sample is placed in freeze drier freeze-drying.
A kind of purifying of Teriparatide acetate provided by the invention and ionic control method, compared with the prior art have with
Lower advantage：Primary purifying and secondarily purified control impurity are carried out by using high pressure reverse-phase chromatography, and hydrochloric acid is added after turning salt
Carry out acetate plasma content control is combined, repeatedly purify and turn salt process use different chromatographic systems, scale system
Standby (a batch can obtain 200 grams of finished product or more) purity and ion concentration meet the Teriparatide acetate of quality requirement in USP 40
Bulk pharmaceutical chemicals, obtained Teriparatide purity are more than 99%, methionine sulfoxide Teriparatide impurity summation≤0.50% and it is maximum not
Know it is single it is miscellaneous≤0.10%, acetate ion content≤5%, chloride ion content≤4%, trifluoroacetic acid root and sulfate radical content≤
0.1%, purifying total recovery is more than 60%.
Description of the drawings
It is that the chromatography spectrogram and HPLC spectrograms of Teriparatide acetate are obtained using embodiment three below.
Fig. 1 is the 1st purifying chromatography spectrogram in embodiment one.
Fig. 2 is the target peak HPLC spectrograms collected after the 1st chromatography in embodiment one.
Fig. 3 is the 2nd purifying chromatography spectrogram in embodiment one.
Fig. 4 is the 2nd target peak HPLC spectrogram collected after purification in embodiment one.
Fig. 5 is the chromatography of ions figure that sample is directly lyophilized after two transfer salt of embodiment.
Fig. 6 is the chromatography of ions figure of addition hydrochloric acid freeze-drying sample after two transfer salt of embodiment.
Specific implementation mode
Embodiment one：
1. sample treatment：The thick peptide of every gram of Teriparatide is dissolved in 50ml volume ratios：Acetonitrile：Water=5:95 acetonitrile solution
In, for ultrasound to being completely dissolved, it is for use to collect filtrate for 0.45 μm of membrane filtration.
2. the 1st purifying：
Purification condition：Chromatographic column：Using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured columns of stationary phase,
Pillar diameter and filling length are：20*25cm.Mobile phase A：Molar concentration is the ammonium bicarbonate aqueous solution of 0.1mol/L, ammonium hydroxide
It is 8.0 to adjust pH；B phases：Acetonitrile.Flow velocity：1200ml/min.Check wavelength 220nm.Gradient：B%：10~30%, 45min, sample introduction
Amount is 80g.
Purification process：Loading after chromatographic column is balanced with mobile phase A, applied sample amount 4L sample solutions.Linear gradient elution
45min collects target peak.The purity using the related substance detecting method detection of Teriparatide in USP 40 of collection is more than
Maximum single miscellaneous≤0.10% target peptide solution is diluted with water one times at 95% and relative retention time RRT 0.9~1.1, waits for
With.Target peptide components recoveries 82%.1st purifying chromatography spectrogram is as shown in Figure 1, the target peak HPLC spectrograms collected after chromatography
As shown in Figure 2.
3. the 2nd purifying：
Purification condition：Chromatographic column：Using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured columns of stationary phase,
Pillar diameter and filling length are：20*25cm.Mobile phase A：Molar concentration is the aqueous sodium persulfate solution of 0.1mol/L, sulfuric acid tune
PH is 3.0；B phases：Acetonitrile.Flow velocity：1200ml/min.Check wavelength 220nm.Gradient：B%：10%~30%, 45min is eluted,
Sample size is 80g.
Purification process：By chromatographic column loading after mobile phase A balance 1CV, applied sample amount 4L sample solutions.Linear gradient elution
45min collects target peak.The purity using the related substance detecting method detection of Teriparatide in USP 40 of collection is more than
99.5%, methionine sulfoxide Teriparatide impurity summation≤0.50% and maximum unknown list it is miscellaneous≤0.10% target peptide solution adds
Water dilutes one times, for use.Target peptide components recoveries 84%.What the 2nd purifying was collected after chromatographing spectrogram as shown in figure 3, chromatographing
Target peak HPLC spectrograms are as shown in Figure 4.
4. turn salt and ionic control
Turn salt condition：Chromatographic column：Using eight alkyl silane bonded silica gels as the DAC-20 dynamic axial pressured columns of stationary phase, column
Sub- diameter and filling length are：20*25cm.Mobile phase A：Molar concentration is the ammonium acetate aqueous solution of 0.02mol/L；B phases：Second
Nitrile.Flow velocity：1200ml/min.Check wavelength 220nm.Sample size is 80g.
Turn salt process：Loading after 1CV after chromatographic column is balanced with mobile phase A, applied sample amount 4L sample solutions.Gradient is set
The gradient isocratic elution 15min of 5%B phases；Then it is 50% acetonitrile solution elution samples of 0.1% acetic acid to use mass concentration,
It is concentrated under reduced pressure into 10~20mg/ml at 35 DEG C of the target peptide solution of collection, 3 equivalent 1N hydrochloric acid are added, for use.Target peptide components return
Yield 97%.
5. freeze-drying
Target peptide solution obtained by upper step is transferred in sizeable stainless steel pallet, purity is obtained after freeze-drying
99.5%, the teriparatide acetate of methionine sulfoxide Teriparatide impurity summation 0.38% and maximum unknown list miscellaneous 0.06%,
Acetate ion content 3.9%, chloride ion content 3.6%, trifluoroacetic acid radical content 0.09% and sulfate radical content 0.08% are pure
It is about 66.8% to change total recovery.
Embodiment two：
1. sample treatment：The thick peptide of every gram of Teriparatide is dissolved in 50ml volume ratios：Acetonitrile：Water=5:95 acetonitrile solution
In, for ultrasound to being completely dissolved, it is for use to collect filtrate for 0.45 μm of membrane filtration.
2. the 1st purifying：
Purification condition：Chromatographic column：Using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured columns of stationary phase,
Pillar diameter and filling length are：20*25cm.Mobile phase A：Molar concentration is the ammonium bicarbonate aqueous solution of 0.01mol/L, acetic acid
It is 7.0 to adjust pH；B phases：Acetonitrile.Flow velocity：1200ml/min.Check wavelength 220nm.Gradient：B%：10%~30%, 45min, into
Sample amount is 80g.
Purification process：Loading after chromatographic column is balanced with mobile phase A, applied sample amount 4L sample solutions.Linear gradient elution
45min collects target peak.The purity using the related substance detecting method detection of Teriparatide in USP 40 of collection is more than
Maximum single miscellaneous≤0.10% target peptide solution is diluted with water one times at 95% and relative retention time RRT 0.9~1.1, waits for
With.Target peptide components recoveries 80%.
3. the 2nd purifying：
Purification condition：Chromatographic column：Using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured columns of stationary phase,
Pillar diameter and filling length are：20*25cm.Mobile phase A：Molar concentration is the aqueous sodium persulfate solution of 0.05mol/L, sulfuric acid tune
PH is 2.0；B phases：Acetonitrile.Flow velocity：1200ml/min.Check wavelength 220nm.Gradient：B%：10%~30%, 45min is eluted,
Sample size is 80g.
Purification process：By chromatographic column loading after mobile phase A balance 1CV, applied sample amount 4L sample solutions.Linear gradient elution
45min collects target peak.The purity using the related substance detecting method detection of Teriparatide in USP 40 of collection is more than
99%, methionine sulfoxide Teriparatide impurity summation≤0.50% and maximum unknown list it is miscellaneous≤0.10% target peptide solution adds water
One times of dilution, for use.Target peptide components recoveries 80%.
4. turn salt and ionic control
Turn salt condition：Chromatographic column：Using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured columns of stationary phase,
Pillar diameter and filling length are：20*25cm.Mobile phase A：Molar concentration is the ammonium acetate aqueous solution of 0.02mol/L；B phases：Second
Nitrile.Flow velocity：1200ml/min.Check wavelength 220nm.Sample size is 80g.
Turn salt process：Loading after 1CV after chromatographic column is balanced with mobile phase A, applied sample amount 4L sample solutions.Gradient is set
The gradient isocratic elution 15min of 5%B phases；Then it is 50% acetonitrile solution elution samples of 0.1% acetic acid to use mass concentration,
It is concentrated under reduced pressure into 10~20mg/ml at 35 DEG C of the target peptide solution of collection, takes half that 1.0 equivalent 1N hydrochloric acid are added, for use.Target
The peptide components rate of recovery 95%.
5. freeze-drying
The target peptide that 1.0 equivalent 1N hydrochloric acid of 1.0 equivalent 1N hydrochloric acid target peptide solutions and addition are not added obtained by upper step is molten
Liquid is transferred to respectively in sizeable stainless steel pallet, and purity is obtained after the two freeze-drying and is all higher than 99%, methionine is sub-
Sulfone Teriparatide impurity summation≤0.50% and maximum unknown list it is miscellaneous≤0.10% teriparatide acetate, chloride ion content≤
4% ,≤0.1%, purifying total recovery is about 60.8% for trifluoroacetic acid root and sulfate radical content.Difference lies in the former acetate from
Sub- content 8%, the latter's acetate ion content 4%, the effect that hydrochloric acid is added herein are to control the content of acetate ion.Turn salt
Directly as shown in figure 5, the peak of serial number 1 is acetate ion in figure, the peak of serial number 2 is the chromatography of ions figure of freeze-drying sample afterwards
The peak of chlorion, serial number 5 is sulfate ion；Turn the chromatography of ions figure of freeze-drying sample after addition hydrochloric acid after salt as shown in fig. 6,
The peak of serial number 2 is acetate ion in figure, and the peak of serial number 4 is chlorion, and the peak of serial number 7 is sulfate ion.
Embodiment three：
1. sample treatment：The thick peptide of every gram of Teriparatide is dissolved in 50ml volume ratios：Acetonitrile：Water=5:95 acetonitrile solution
In, for ultrasound to being completely dissolved, it is for use to collect filtrate for 0.45 μm of membrane filtration.
2. the 1st purifying：
Purification condition：Chromatographic column：Using eight alkyl silane bonded silica gels as the DAC-20 dynamic axial pressured columns of stationary phase, column
Sub- diameter and filling length are：20*25cm.Mobile phase A：Molar concentration is the ammonium bicarbonate aqueous solution of 0.01mol/L, ammonium hydroxide tune
PH is 7.5；B phases：Acetonitrile.Flow velocity：1200ml/min.Check wavelength 220nm.Gradient：B%：10%~30%, 45min, sample introduction
Amount is 80g.
Purification process：Loading after chromatographic column is balanced with mobile phase A, applied sample amount 4L sample solutions.Linear gradient elution
45min collects target peak.The purity using the related substance detecting method detection of Teriparatide in USP 40 of collection is more than
Maximum single miscellaneous≤0.10% target peptide solution is diluted with water one times at 95% and relative retention time RRT 0.9~1.1, waits for
With.Target peptide components recoveries 81%.
3. the 2nd purifying：
Purification condition：Chromatographic column：Using eight alkyl silane bonded silica gels as the DAC-20 dynamic axial pressured columns of stationary phase, column
Sub- diameter and filling length are：20*25cm.Mobile phase A：Molar concentration is the aqueous sodium persulfate solution of 0.05mol/L, sulfuric acid tune pH
It is 2.0；B phases：Acetonitrile.Flow velocity：1200ml/min.Check wavelength 220nm.Gradient：B%：10~30%, elute 45min, sample introduction
Amount is 80g.
Purification process：By chromatographic column loading after mobile phase A balance 1CV, applied sample amount 4L sample solutions.Linear gradient elution
45min collects target peak.The purity using the related substance detecting method detection of Teriparatide in USP 40 of collection is more than
99%, methionine sulfoxide Teriparatide impurity summation≤0.50% and maximum unknown list it is miscellaneous≤0.10% target peptide solution adds water
One times of dilution, for use.Target peptide components recoveries 82%.
4. turn salt and ionic control
Turn salt condition：Chromatographic column：Using eight alkyl silane bonded silica gels as the DAC-20 dynamic axial pressured columns of stationary phase, column
Sub- diameter and filling length are：20*25cm.Mobile phase A：Molar concentration is the ammonium acetate aqueous solution of 0.02mol/L；B phases：Second
Nitrile.Flow velocity：1200ml/min.Check wavelength 220nm.Sample size is 80g.
Turn salt process：Loading after 1CV after chromatographic column is balanced with mobile phase A, applied sample amount 4L sample solutions.Gradient is set
The gradient isocratic elution 15min of 5%B phases；Then it is 50% acetonitrile solution elution samples of 0.1% acetic acid to use mass concentration,
It is concentrated under reduced pressure into 10~20mg/ml at 35 DEG C of the target peptide solution of collection, 2.0 equivalent 1N hydrochloric acid are added, for use.Target peptide components
The rate of recovery 94%.
5. freeze-drying
Target peptide solution obtained by upper step is transferred in sizeable stainless steel pallet, purity is obtained after freeze-drying
99.2%, the teriparatide acetate of methionine sulfoxide Teriparatide impurity summation 0.48% and maximum unknown list miscellaneous 0.08%,
Acetate ion content 4.8%, chloride ion content 3.5%, trifluoroacetic acid radical content 0.06%, sulfate radical content 0.08%%,
It is about 62.4% to purify total recovery.
Example IV：
1. sample treatment：The thick peptide of every gram of Teriparatide is dissolved in 50ml volume ratios：Acetonitrile：Water=5:95 acetonitrile solution
In, for ultrasound to being completely dissolved, it is for use to collect filtrate for 0.45 μm of membrane filtration.
2. the 1st purifying：
Purification condition：Chromatographic column：Using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured columns of stationary phase,
Pillar diameter and filling length are：20*25cm.Mobile phase A：Molar concentration is the ammonium bicarbonate aqueous solution of 0.2mol/L, ammonium hydroxide
It is 9.0 to adjust pH；B phases：Acetonitrile.Flow velocity：1200ml/min.Check wavelength 220nm.Gradient：B%：10%~30%, 45min, into
Sample amount is 80g.
Purification process：Loading after chromatographic column is balanced with mobile phase A, applied sample amount 4L sample solutions.Linear gradient elution
45min collects target peak.The purity using the related substance detecting method detection of Teriparatide in USP 40 of collection is more than
Maximum single miscellaneous≤0.10% target peptide solution is diluted with water one times at 95% and relative retention time RRT 0.9~1.1, waits for
With.Target peptide components recoveries 83%.
3. the 2nd purifying：
Purification condition：Chromatographic column：Using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured columns of stationary phase,
Pillar diameter and filling length are：20*25cm.Mobile phase A：Molar concentration is the aqueous sodium persulfate solution of 0.2mol/L, sulfuric acid tune
PH is 3.0；B phases：Acetonitrile.Flow velocity：1200ml/min.Check wavelength 220nm.Gradient：B%：10%~30%, 45min is eluted,
Sample size is 80g.
Purification process：By chromatographic column loading after mobile phase A balance 1CV, applied sample amount 4L sample solutions.Linear gradient elution
45min collects target peak.The purity using the related substance detecting method detection of Teriparatide in USP 40 of collection is more than
99%, methionine sulfoxide Teriparatide impurity summation≤0.50% and maximum unknown list it is miscellaneous≤0.10% target peptide solution adds water
One times of dilution, for use.Target peptide components recoveries 80%.
4. turn salt and ionic control
Turn salt condition：Chromatographic column：Using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured columns of stationary phase,
Pillar diameter and filling length are：20*25cm.Mobile phase A：Molar concentration is the ammonium acetate aqueous solution of 0.02mol/L；B phases：Second
Nitrile.Flow velocity：1200ml/min.Check wavelength 220nm.Sample size is 80g.
Turn salt process：Loading after 1CV after chromatographic column is balanced with mobile phase A, applied sample amount 4L sample solutions.Gradient is set
The gradient isocratic elution 15min of 5%B phases；Then it is 50% acetonitrile solution elution samples of 0.1% acetic acid to use mass concentration,
It is concentrated under reduced pressure into 10~20mg/ml at 35 DEG C of the target peptide solution of collection, 4.0 equivalent 1N hydrochloric acid are added, for use.Target peptide components
The rate of recovery 91%.
5. freeze-drying
Target peptide solution obtained by upper step is transferred in sizeable stainless steel pallet, purity is obtained after freeze-drying
99.1%, the teriparatide acetate of methionine sulfoxide Teriparatide impurity summation 0.44% and maximum unknown list miscellaneous 0.09%,
Acetate ion content 4.2%, chloride ion content 3%, trifluoroacetic acid radical content 0.07% and sulfate radical content equal 0.05% are pure
It is about 60.4% to change total recovery.
Embodiment five：
1. sample treatment：The thick peptide of every gram of Teriparatide is dissolved in 50ml volume ratios：Acetonitrile：Water=5:95 acetonitrile solution
In, for ultrasound to being completely dissolved, it is for use to collect filtrate for 0.45 μm of membrane filtration.
2. the 1st purifying：
Purification condition：Chromatographic column：Using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured columns of stationary phase,
Pillar diameter and filling length are：20*25cm.Mobile phase A：Molar concentration is the ammonium bicarbonate aqueous solution of 0.01mol/L, ammonium hydroxide
It is 9.5 to adjust pH；B phases：Acetonitrile.Flow velocity：1200ml/min.Check wavelength 220nm.Gradient：B%：10%~30%, 45min, into
Sample amount is 80g.
Purification process：Loading after chromatographic column is balanced with mobile phase A, applied sample amount 4L sample solutions.Linear gradient elution
45min collects target peak.The purity using the related substance detecting method detection of Teriparatide in USP 40 of collection is more than
Maximum single miscellaneous≤0.10% target peptide solution is diluted with water one times at 95% and relative retention time RRT 0.9~1.1, waits for
With.Target peptide components recoveries 80%.
3. the 2nd purifying：
Purification condition：Chromatographic column：Using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured columns of stationary phase,
Pillar diameter and filling length are：20*25cm.Mobile phase A：Molar concentration is the aqueous sodium persulfate solution of 0.05mol/L, sulfuric acid tune
PH is 4.0；B phases：Acetonitrile.Flow velocity：1200ml/min.Check wavelength 220nm.Gradient：B%：10~30%, 45min is eluted, into
Sample amount is 80g.
Purification process：By chromatographic column loading after mobile phase A balance 1CV, applied sample amount 4L sample solutions.Linear gradient elution
45min collects target peak.The purity using the related substance detecting method detection of Teriparatide in USP 40 of collection is more than
99%, methionine sulfoxide Teriparatide impurity summation≤0.50% and maximum unknown list it is miscellaneous≤0.10% target peptide solution adds water
One times of dilution, for use.Target peptide components recoveries 81%.
4. turn salt and ionic control
Turn salt condition：Chromatographic column：Using octadecylsilane chemically bonded silica as the DAC-20 dynamic axial pressured columns of stationary phase,
Pillar diameter and filling length are：20*25cm.Mobile phase A：Molar concentration is the ammonium acetate aqueous solution of 0.02mol/L；B phases：Second
Nitrile.Flow velocity：1200ml/min.Check wavelength 220nm.Sample size is 80g.
Turn salt process：Loading after 1CV after chromatographic column is balanced with mobile phase A, applied sample amount 4L sample solutions.Gradient is set
The gradient isocratic elution 15min of 5%B phases；Then it is 50% acetonitrile solution elution samples of 0.1% acetic acid to use mass concentration,
It is concentrated under reduced pressure into 10~20mg/ml at 35 DEG C of the target peptide solution of collection, 3.5 equivalent 1N hydrochloric acid are added, for use.Target peptide components
The rate of recovery 94%.
5. freeze-drying
Target peptide solution obtained by upper step is transferred in sizeable stainless steel pallet, purity is obtained after freeze-drying
99.3%, the teriparatide acetate of methionine sulfoxide Teriparatide impurity summation 0.5% and maximum unknown list miscellaneous 0.10%, vinegar
Acid ion content 5%, chloride ion content 4%, trifluoroacetic acid root and sulfate radical content equal 0.1%, purifying total recovery are about
60.9%.
The related substance of the teriparatide acetate obtained by technique productions using embodiment one to embodiment five and ion concentration
Meet the requirements of USP 40, other indices also meet USP 40, while also being compliant with the requirements of EP 9.0, can be used as acetic acid spy
Vertical pa peptide bulk pharmaceutical chemicals export US and European, while can also meet Chinese market requirements of customs declaration.
Comparative example one：
The mobile phase A used is purified by the 1st time in embodiment three and replaces with ammonium acetate buffer solution, with acetic acid or ammonium hydroxide tune
PH, passes through secondarily purified, finally turns also to use HCl treatment after salt, remaining technique and each parameter are the same as embodiment three.
Obtain purity 90.0% after final freeze-drying, methionine sulfoxide Teriparatide impurity summation 0.62% and it is maximum not
Know single miscellaneous 0.15% teriparatide acetate, acetate ion content 3.9%, chloride ion content 4.8%, trifluoroacetic acid root contains
Amount 0.18% and sulfate radical content 0.20%, purifying total recovery is about 60.2%.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
The specific implementation of the present invention is confined to these explanations.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, several simple deductions and replacement can also be made, all shall be regarded as belonging to the present invention's
Protection domain.
Claims (10)
1. purifying and the ionic control method of a kind of Teriparatide acetate, it is characterised in that include the following steps：
Step 1)：Thick peptide solution is subjected to first time gradient elution purifying using chromatographic column, collects eluent；The chromatographic column makes
Mobile phase includes mobile phase A and Mobile phase B, and the mobile phase A is ammonium bicarbonate aqueous solution, with acetic acid or ammonium hydroxide tune pH,
The Mobile phase B is acetonitrile；
Step 2)：The eluent of above-mentioned collection is subjected to second of gradient elution purifying using chromatographic column, collects eluent；It is described
The mobile phase that chromatographic column uses includes mobile phase A and Mobile phase B, and the mobile phase A is aqueous sodium persulfate solution, with sulfuric acid tune pH,
The Mobile phase B is acetonitrile；
Step 3)：The eluent collected in step 2) is subjected to efficient liquid phase using chromatographic column and turns salt, wherein first using ammonium acetate water
Dissolved in acetonitrile system elutes, then is eluted with aqueous acetic acid acetonitrile system；
Step 4)：Hydrochloric acid is added after eluent after step 3) transfer salt is concentrated under reduced pressure, the residual quantity of acetate is made to control
5% or less.
2. according to the method described in claim 1, it is characterized in that：The pH of mobile phase A is 7.0~9.5 in the step 1),
A concentration of 0.01~0.2mol/L of ammonium hydrogen carbonate.
3. according to the method described in claim 2, it is characterized in that：The pH of mobile phase A is 8.0 in the step 1), carbonic acid
A concentration of 0.1mol/L of hydrogen ammonium.
4. according to the method described in claim 1, it is characterized in that：The pH of mobile phase A is 2.0~4.0 in the step 2),
A concentration of 0.05~0.2mol/L of sodium sulphate.
5. according to the method described in claim 4, it is characterized in that：The pH of mobile phase A is 3.0 in the step 2), sulfuric acid
A concentration of 0.1mol/L of sodium.
6. according to the method described in claim 1, it is characterized in that：Hydrochloric acid used in acetic acid radical content is adjusted in the step 4)
Amount is 1~4N.
7. according to the method described in claim 6, it is characterized in that：Hydrochloric acid used in acetic acid radical content is adjusted in the step 4)
Amount is 3.3N.
8. according to the method described in claim 6, it is characterized in that：The mesh of 1~4 equivalent 1N hydrochloric acid will be added in the step 4)
Mark peptide solution, which is transferred in sizeable stainless steel pallet, to be freeze-dried, and obtained Teriparatide purity is more than 99%,
Methionine sulfoxide Teriparatide impurity summation≤0.50% and maximum unknown list it is miscellaneous≤0.10% teriparatide acetate, acetate
≤ 0.1%, purifying yield is 60% for ion concentration≤5%, chloride ion content≤4%, trifluoroacetic acid root and sulfate radical content.
9. according to claim 1~8 any one of them method, it is characterised in that：Step 1) to chromatographic column described in step 3) makes
Stationary phase is octadecylsilane chemically bonded silica or eight alkyl silane bonded silica gels.
10. according to claim 1~8 any one of them method, it is characterised in that：Further comprise the steps：Above-mentioned
Following sample treatment is carried out before step 1), by thick peptide using volume ratio 5~10% acetonitrile solution according to concentration 5~
20g/L dissolves, and then uses 0.22~0.45 μm of membrane filtration, it is for use to collect filtrate.
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| CN109897099A (en) * | 2019-03-27 | 2019-06-18 | 哈尔滨吉象隆生物技术有限公司 | A kind of preparation method of Teriparatide |
| CN111057142A (en) * | 2018-10-17 | 2020-04-24 | 深圳市健元医药科技有限公司 | Purification method of teriparatide |
| CN112940102A (en) * | 2020-12-30 | 2021-06-11 | 苏州天马医药集团天吉生物制药有限公司 | Purification method of Somalutide |
| CN114478750A (en) * | 2021-12-28 | 2022-05-13 | 深圳翰宇药业股份有限公司 | Purification method of teriparatide |
| CN115656391A (en) * | 2022-12-12 | 2023-01-31 | 哈尔滨吉象隆生物技术有限公司 | Method for detecting impurities contained in teriparatide |
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| CN111057142A (en) * | 2018-10-17 | 2020-04-24 | 深圳市健元医药科技有限公司 | Purification method of teriparatide |
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| CN112940102A (en) * | 2020-12-30 | 2021-06-11 | 苏州天马医药集团天吉生物制药有限公司 | Purification method of Somalutide |
| CN114478750A (en) * | 2021-12-28 | 2022-05-13 | 深圳翰宇药业股份有限公司 | Purification method of teriparatide |
| CN114478750B (en) * | 2021-12-28 | 2024-04-02 | 深圳翰宇药业股份有限公司 | Purification method of teriparatide |
| CN115656391A (en) * | 2022-12-12 | 2023-01-31 | 哈尔滨吉象隆生物技术有限公司 | Method for detecting impurities contained in teriparatide |
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