CN102731643A - Method for preparing polypeptide used for treating osteoporosis - Google Patents
Method for preparing polypeptide used for treating osteoporosis Download PDFInfo
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- CN102731643A CN102731643A CN2012102130443A CN201210213044A CN102731643A CN 102731643 A CN102731643 A CN 102731643A CN 2012102130443 A CN2012102130443 A CN 2012102130443A CN 201210213044 A CN201210213044 A CN 201210213044A CN 102731643 A CN102731643 A CN 102731643A
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Abstract
The invention belongs to the pharmacochemistry technical field, and discloses a method for preparing polypeptide used for treating osteoporosis, concretely relates to a teriparatide preparation method. The preparation method is characterized by comprising the following steps: preparing each peptide resin fragment for forming teriparatide, gradually coupling each peptide resin fragment to teriparatide on a solid phase, then pyrolysizing to obtain the teriparatide crude product, and purifying to obtain the product teriparatide. Compared with the prior art, the preparation method has the advantages of simple operation, short synthesis cycle, low cost, less environmental pollution and high yield of teriparatide, and the total yield is 30%. The method of the invention is suitable for large-scale industrial production of teriparatide, and the prepared teriparatide has the advantages of high purity and less by-products, and has considerable economical and practical value as well as wide application prospect.
Description
Technical field
The invention belongs to the pharmaceutical chemistry technical field, relate to a kind of preparation method who treats the osteoporosis polypeptide.Relate in particular to a kind of preparation method of teriparatide.
Background technology
Teriparatide, English Teriparatide by name is the 1-34 amino acid fragment that contains the N-stub area of 84 amino acid whose endogenous parathyroid hormone PTH biologically actives, molecular formula is C
181H
291N
55O
51S
2, molecular weight is 4177.77, its peptide preface is:
1 2 3 4 ?5 ?6 ?7 ?8 ?9 10 11 12 13 14 15 16 17 ?18 19 20H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-21 22 ?23?24 25 26 ?27?28 29 30 ?31?32 ?33 34Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH。
Teriparatide is developed by Lilly Co., Eli.; U.S. food Drug Administration has ratified the osteoporosis with height risk of bone fracture that teriparatide is used to treat man and menopausal women on November 26th, 2002, also be used to treat former of the man with or because the osteoporosis that sexual hypofunction causes with high risk of bone fracture.Teriparatide is first kind of bone forming agent kind new medicine that obtains U.S. food and FAD FDA approval.Present conventional medicine for treating osteoporosis generally just acts on osteoclast and slows down or block bone-loss, and the verivate of this Rat parathyroid hormone 1-34 of teriparatide has and increases the scleroblast number, strengthens its effect active and the prevention TNF-a Induced Apoptosis in Osteoblasts.Because of it can increase osteoblastic activity and quantity, thus can promote osteogenesis, be used for serious osteoporotic second line treatment.The teriparatide spinoff is very little in addition, is nauseating, dizzy and leg cramps usually.Therefore teriparatide has very high pharmaceutical use and vast market prospect.
Gift comes company to adopt genetic expression to obtain teriparatide.Also have many pieces of genetic expressions to obtain the patent such as the US6590081 of teriparatide in addition, however genetic expression has that workload is big, serious three wastes, can't scale operation etc. shortcoming.
At present, also having a kind of popular method is progressively synthetic (SPPS) method of solid phase, and this method has characteristics such as simple to operate, that equipment requirements is low.But because teriparatide is made up of 34 amino acid, progressively the compound method product yield is low for solid phase, and the purifying products difficulty, is difficult to obtain highly purified teriparatide.
Summary of the invention
In view of this, the objective of the invention is the defective to prior art, the preparation method of the high teriparatide of a kind of yield is provided, the teriparatide purity that said method makes is high, is suitable for the scale operation of teriparatide.
For realizing the object of the invention, the present invention adopts following technical scheme:
A kind of preparation method of teriparatide, each peptide resin fragment of teriparatide is formed in preparation, with each peptide resin fragment on solid phase progressively coupling obtain the teriparatide resin, cracking obtains the teriparatide bullion then, purifying obtains the product teriparatide.
The preparation method of teriparatide according to the invention adopts different fragments coupling strategy; Each peptide resin fragment of forming teriparatide through solid phase synthesis; Be carrier then with the appropriate resin; With each peptide resin fragment on solid phase progressively coupling obtain the teriparatide resin, last cracking obtains the teriparatide bullion, purifying obtains the product teriparatide.Segmental synthetic can the carrying out simultaneously of each peptide resin of composition teriparatide according to the invention makes synthesis cycle reduce 2/3, and midbody is prone to purifying; Cost is low, finally prepares teriparatide product purity height, and by product is few; Product yield is high, is beneficial to the scale operation of teriparatide.
Teriparatide is made up of 34 amino-acid residues, therefore has the possible fragment and the coupling order of magnanimity.In an embodiment; 34 amino-acid residues forming teriparatide are divided into 5 fragments, i.e. teriparatide sequence 1-6 amino acids, sequence 7-12 amino acids, sequence 13-20 amino acids, sequence 21-25 amino acids, sequence 26-34 amino acids.Said preparation method specifically may further comprise the steps:
A) 5 peptide fragment of teriparatide are formed in preparation: peptide resin fragment 1, side chain protected peptide fragment 2; Side chain protected peptide fragment 3, side chain protected peptide fragment 4 and side chain protected peptide fragment 5; Wherein said peptide resin fragment 1 is a teriparatide sequence 34-26 amino acids; Said side chain protected peptide fragment 2 is a teriparatide sequence 25-21 amino acids; Said side chain protected peptide fragment 3 is a teriparatide sequence 20-13 amino acids, and said side chain protected peptide fragment 4 is a teriparatide sequence 12-7 amino acids, and said side chain protected peptide fragment 5 is a teriparatide sequence 6-1 amino acids;
B) side chain protected peptide fragment 2 and 1 coupling of peptide resin fragment are obtained the peptide resin I;
C) side chain protected peptide fragment 3 and the coupling of peptide resin I are obtained the peptide resin II;
D) side chain protected peptide fragment 4 and the coupling of peptide resin II are obtained the peptide resin III;
E) side chain protected peptide fragment 5 and the coupling of peptide resin III are obtained the peptide resin IV;
F) cracking of peptide resin IV obtains the teriparatide bullion
G) the purified teriparatide that obtains of teriparatide bullion.
Wherein said peptide resin fragment 1 is H-Lys (Boc)-Lys (Boc)-Leu-Gln (Trt)-Asp (otbu)-Val-His (Trt)-Asn (Trt)-Phe-resin.
Said side chain protected peptide fragment 2 is Fmoc-Val-Glu (otbu)-Trp (Boc)-Leu-Arg (pbf)-OH.
Said side chain protected peptide fragment 3 is Fmoc-Lys (Boc)-His (Trt)-Leu-Asn (Trt)-Ser (tbu)-Met-Glu (otbu)-Arg (pbf)-OH.
Said side chain protected peptide fragment 4 is Fmoc-Leu-Met-His (Trt)-Asn (Trt)-Leu-Gly-OH.
Said side chain protected peptide fragment 5 is Fmoc-Ser (tbu)-Val-Ser (tbu)-Glu (otbu)-IIe-Gln (Trt)-OH.
Said side chain protected peptide fragment 2 is H-Val-Glu (otbu)-Trp (Boc)-Leu-Arg (pbf) with the peptide resin fragment I that 1 coupling of peptide resin fragment obtains--Lys (Boc)-Lys (Boc)-Leu-Gln (Trt)-Asp (otbu)-Val-His (Trt)-Asn (Trt)-Phe-resin.
The peptide resin fragment II that said side chain protected peptide fragment 3 and the coupling of peptide resin fragment I obtain--Val-Glu (otbu)-Trp (Boc)-Leu-Arg (the pbf)--Lys (Boc)-Lys (Boc)-Leu-Gln (Trt)-Asp (otbu)-Val-His (Trt)-Asn (Trt)-Phe-resin that is H-Lys (Boc)-His (Trt)-Leu-Asn (Trt)-Ser (tbu)-Met-Glu (otbu)-Arg (pbf).
The peptide resin fragment III that said side chain protected peptide fragment 4 and the coupling of peptide resin fragment II obtain--Val-Glu (otbu)-Trp (Boc)-Leu-Arg (the pbf)--Lys (Boc)-Lys (Boc)-Leu-Gln (Trt)-Asp (otbu)-Val-His (Trt)-Asn (Trt)-Phe-resin that is H-Leu-Met-His (Trt)-Asn (Trt)-Leu-Gly--Lys (Boc)-His (Trt)-Leu-Asn (Trt)-Ser (tbu)-Met-Glu (otbu)-Arg (pbf).
Said side chain protected peptide fragment 5 is H-Ser with the peptide resin fragment IV that the coupling of peptide resin fragment III obtains; (tbu)-Val-Ser; (tbu)-Glu; (otbu)-IIe-Gln; (Trt)--Leu-Met-His; (Trt)-Asn; (Trt)-Leu-Gly--Lys; (Boc)-His; (Trt)-Leu-Asn; (Trt)-Ser; (tbu)-Met-Glu; (otbu)-Arg; (pbf)--Val-Glu; (otbu)-Trp; (Boc)-Leu-Arg; (pbf)--Lys; (Boc)-Lys; (Boc)-Leu-Gln; (Trt)-Asp; (otbu)-Val-His; (Trt)-Asn; (Trt)-the Phe-resin.
As preferably, said steps A) peptide resin fragment 1 used solid phase carrier is king's resin in.
As preferably, said steps A) side chain protected peptide fragment 2, side chain protected peptide fragment 3, side chain protected peptide fragment 4 and side chain protected peptide fragment 5 used solid phase carriers are 2-chlorine trityl chloride resin in.
As preferably, said steps A) coupling agent of peptide resin fragment 1, side chain protected peptide fragment 2, side chain protected peptide fragment 3, side chain protected peptide fragment 4 and side chain protected peptide fragment 5 is HOBT/DIC, PyBOP/HOBt/DIPEA or TBTU/HOBt/DIPEA in.
Side chain protected peptide fragment 2, side chain protected peptide fragment 3, side chain protected peptide fragment 4 and side chain protected peptide fragment 5 are coupled at cracking acquisition in back on the resin with amino acid.As preferably, said steps A) cracking agent of side chain protected peptide fragment 2, side chain protected peptide fragment 3, side chain protected peptide fragment 4, side chain protected peptide fragment 5 is TFE in.
Obtain bullion with ether sedimentation after side chain protected peptide fragment 2, side chain protected peptide fragment 3, side chain protected peptide fragment 4 and side chain protected peptide fragment 5 cracking from resin are got off, bullion need not the direct freeze-drying of purifying, and control moisture is below 2%.Full guard fragment purity after the freeze-drying can directly be used for next step reaction all greater than 90%.
As preferably, said step B)-step e) in side chain protected peptide and peptide resin link coupled coupling agent be HOBT/DIC, PyBOP/HOBt/DIPEA or TBTU/HOBt/DIPEA.
As preferably, said step B)-step e) in side chain protected peptide fragment and peptide resin link coupled reaction solvent be among DMF, DCM, NMP, the DMSO one or more.
That is to say, as step B according to the invention) in, the side chain protected peptide fragment 2 after the freeze-drying obtains the peptide resin I with peptide resin 1 reaction, and the coupling agent of employing is preferably HOBT/DIC, PyBOP/HOBt/DIPEA or TBTU/HOBt/DIPEA.TBTU/HOBt/DIPEA more preferably.Further, the mol ratio of said TBTU, HOBt and DIPEA is 1:1:2.
That is to say, as step B according to the invention) in, the side chain protected peptide fragment 2 after the freeze-drying obtains the peptide resin I with peptide resin 1 reaction, and the reaction solvent of employing is one or more among DMF, DCM, NMP, the DMSO.More have and be preferably DMF and the DMSO volume ratio is the mixed solution of 1:1.
That is to say, as step C according to the invention) in, the side chain protected peptide fragment 3 after the freeze-drying obtains the peptide resin II with the coupling of peptide resin I, and the coupling agent of employing is HOBT/DIC, PyBOP/HOBt/DIPEA or TBTU/HOBt/DIPEA.PyBOP/HOBt/DIPEA more preferably.Further, the mol ratio of said PyBOP, HOBt and DIPEA is 1:1:2.
That is to say, as step C according to the invention) in, the side chain protected peptide fragment 2 after the freeze-drying obtains the peptide resin I with 1 reaction of peptide resin fragment, and the reaction solvent of employing is one or more among DMF, DCM, NMP, the DMSO.More have and be preferably DMF and the NMP volume ratio is the mixed solution of 1:1.
That is to say, as step D according to the invention) in, the side chain protected peptide fragment 4 after the freeze-drying obtains the peptide resin III with the coupling of peptide resin II, and the coupling agent of employing is HOBT/DIC, PyBOP/HOBt/DIPEA or TBTU/HOBt/DIPEA.PyBOP/HOBt/DIPEA more preferably.Further, the mol ratio of said PyBOP, HOBt and DIPEA is 1:1:2.
That is to say, as step D according to the invention) in, the side chain protected peptide fragment 4 after the freeze-drying obtains the peptide resin III with the coupling of peptide resin II, and the reaction solvent of employing is one or more among DMF, DCM, NMP, the DMSO.More have and be preferably DMF and the DMSO volume ratio is the mixed solution of 1:1.
That is to say, as step e according to the invention) in, the side chain protected peptide fragment 5 after the freeze-drying obtains the peptide resin IV with the coupling of peptide resin III, and the coupling agent of employing is HOBT/DIC, PyBOP/HOBt/DIPEA or TBTU/HOBt/DIPEA.PyBOP/HOBt/DIPEA more preferably.Further, the mol ratio of said PyBOP, HOBt and DIPEA is 1:1:2.
That is to say, as step e according to the invention) in, the side chain protected peptide fragment 5 after the freeze-drying obtains the peptide resin IV with the coupling of peptide resin III, and the reaction solvent of employing is one or more among DMF, DCM, NMP, the DMSO.More have and be preferably DMF and the NMP volume ratio is the mixed solution of 1:1.
As preferably; Said step F) in, peptide resin IV cracked lysate is that TFA and water volume ratio are that mixing solutions, TFA, DET, PHOH and the water volume ratio of 95:5 is that mixing solutions or TFA, DET, TIS, PHOH and the water volume ratio of 90:5:3:2 is the mixing solutions of 80:5:5:5:5.Further be preferably the mixing solutions that TFA, DET, TIS, PHOH and water volume ratio are 80:5:5:5:5.
As preferably, said preparing method's step G) purifying is the RPLC purifying in.
RPLC, English name reversed phase high performance liquid chromatography is called for short, RP-HPLC, the liquid chromatography system of being made up of non-polar stationary phase and polarity moving phase.It is just in time opposite with the liquid chromatography system of being made up of polar stationary phase and low-pole moving phase (normal-phase chromatography).RP-HPLC is the topmost clastotype of current liquid chromatography, almost can be used for all and can be dissolved in the organic separation and purification in polarity or the weak polar solvent.Step G according to the invention) adopt RPLC purifying, freeze-drying to obtain teriparatide, purity is greater than 99%.
Further; In a certain embodiment, the chromatographic condition of said RPLC purifying is: adopt Waters 2695RP-HPLC system, wavelength 230nm; Chromatographic column is 50 * 250mm anti-phase C18 post; Rule 0.2%TFA (A)-acetonitrile (B) is the moving phase purifying, collects purpose peak cut, obtains purity greater than 98.5% smart peptide.Smart peptide solution is adopted Waters 2695RP-HPLC system, and chromatographic column is 50 * 250mm anti-phase C18 post, and 0.2% acetum (A)-acetonitrile (B) changes salt for moving phase, collects purpose peak cut, and rotary evaporation concentrates, and freeze-drying obtains the smart peptide of teriparatide acetate salt.
In some specific embodiments, 34 amino-acid residues that the present invention will form teriparatide are divided into 3 fragments, i.e. teriparatide sequence 1-10 amino acids, sequence 11-25 amino acids and sequence 26-34 amino acids.Said preparation method specifically may further comprise the steps:
1) 3 peptide fragment of teriparatide are formed in preparation: peptide resin fragment a, side chain protected peptide fragment b and side chain protected peptide fragment c; Wherein said peptide resin fragment a is a teriparatide sequence 34-26 amino acids; Said side chain protected peptide fragment b is a teriparatide sequence 25-11 amino acids, and said side chain protected peptide fragment c is a teriparatide sequence 10-1 amino acids;
2) side chain protected peptide fragment b and peptide resin fragment a coupling are obtained peptide resin A;
3) side chain protected peptide fragment 3 and peptide resin A coupling are obtained peptide resin B;
4) peptide resin B cracking obtains the teriparatide bullion
5) the purified teriparatide that obtains of teriparatide bullion.
In some specific embodiments, 34 amino-acid residues that the present invention will form teriparatide are divided into 4 fragments, i.e. teriparatide sequence 1-6 amino acids, sequence 7-15 amino acids, sequence 16-28 amino acids and sequence 29-34 amino acids.Said preparation method specifically may further comprise the steps:
A) 4 peptide fragment of teriparatide are formed in preparation: peptide resin Segment A, side chain protected peptide fragment B; Side chain protected peptide fragment C and side chain protected peptide fragment D; Wherein said peptide resin Segment A is a teriparatide sequence 34-29 amino acids; Said side chain protected peptide fragment B is a teriparatide sequence 28-16 amino acids, and said side chain protected peptide fragment C is a teriparatide sequence 15-7 amino acids, and said side chain protected peptide fragment D is a teriparatide sequence 6-1 amino acids;
B) side chain protected peptide fragment B and the coupling of peptide resin Segment A are obtained peptide resin i;
C) side chain protected peptide fragment C and peptide resin i coupling are obtained peptide resin ii;
D) side chain protected peptide fragment D and peptide resin ii coupling are obtained peptide resin iii;
E) peptide resin iii cracking obtains the teriparatide bullion
F) the purified teriparatide that obtains of teriparatide bullion.
In some specific embodiments; 34 amino-acid residues that the present invention will form teriparatide are divided into 5 fragments, i.e. teriparatide sequence 1-6 amino acids, sequence 7-12 amino acids, sequence 13-19 amino acids, sequence 20-28 amino acids, sequence 29-34 amino acids.The preparation method specifically may further comprise the steps:
I) 5 peptide fragment of teriparatide are formed in preparation: the peptide resin fragment 1., the side chain protected peptide fragment 2.; The side chain protected peptide fragment 3., the side chain protected peptide fragment 4. with the side chain protected peptide fragment 5.; 1. wherein said peptide resin fragment is teriparatide sequence 34-29 amino acids; 2. said side chain protected peptide fragment is teriparatide sequence 28-20 amino acids; 3. said side chain protected peptide fragment is teriparatide sequence 19-13 amino acids, and 4. said side chain protected peptide fragment is teriparatide sequence 12-7 amino acids, and 5. said side chain protected peptide fragment is teriparatide sequence 6-1 amino acids;
II) side chain protected peptide fragment 2 and 1 coupling of peptide resin fragment are obtained peptide resin a;
III) side chain protected peptide fragment 3 and peptide resin a coupling are obtained peptide resin b;
IV) side chain protected peptide fragment 4 and peptide resin b coupling are obtained peptide resin c;
V) side chain protected peptide fragment 5 and peptide resin c coupling are obtained peptide resin d;
VI) peptide resin d cracking obtains the teriparatide bullion;
VII) the purified teriparatide that obtains of teriparatide bullion.
In other specific embodiments; 34 amino-acid residues that the present invention will form teriparatide are divided into 5 fragments, i.e. teriparatide sequence 1-6 amino acids, sequence 7-12 amino acids, sequence 13-20 amino acids, sequence 21-28 amino acids, sequence 29-34 amino acids.Said preparation method specifically may further comprise the steps:
I) 5 peptide fragment of teriparatide are formed in preparation: peptide resin fragment I, side chain protected peptide fragment II; Side chain protected peptide fragment III, side chain protected peptide fragment IV and side chain protected peptide fragment V; Wherein said peptide resin fragment I is a teriparatide sequence 34-29 amino acids; Said side chain protected peptide fragment II is a teriparatide sequence 28-21 amino acids; Said side chain protected peptide fragment III is a teriparatide sequence 20-13 amino acids, and said side chain protected peptide fragment IV is a teriparatide sequence 12-7 amino acids, and said side chain protected peptide fragment V is a teriparatide sequence 6-1 amino acids;
Ii) 1. side chain protected peptide fragment II and the coupling of peptide resin fragment I are obtained peptide resin;
Iii) with side chain protected peptide fragment III and peptide resin 1. coupling obtain peptide resin 2.;
Iv) with side chain protected peptide fragment IV and peptide resin 2. coupling obtain peptide resin 3.;
V) with side chain protected peptide fragment V and peptide resin 3. coupling obtain peptide resin 4.;
Vi) peptide resin 4. cracking obtain the teriparatide bullion
The vii) purified teriparatide that obtains of teriparatide bullion.
Compared with prior art; Preparing method according to the invention is simple to operate, synthesis cycle is short, cost is low, environmental pollution is little, the teriparatide yield high; Total recovery is suitable for the teriparatide large-scale industrialized production greater than 30%, and the smart peptide purity of the teriparatide acetate that makes is greater than 99%; Purity is high, by product is few, has considerable economical and practical value and application prospects.
Embodiment
The embodiment of the invention discloses a kind of preparation method of teriparatide.Those skilled in the art can use for reference this paper content, suitably improve processing parameter and realize.Special needs to be pointed out is that all similarly replace and change apparent to those skilled in the art, they all are regarded as and are included in the present invention.Method of the present invention is described through preferred embodiment, and the related personnel obviously can change or suitably change and combination method as herein described in not breaking away from content of the present invention, spirit and scope, realizes and use technology of the present invention.
In order further to understand the present invention, the present invention is elaborated below in conjunction with embodiment.
During the implication of employed abbreviation is listed in the table below in specification sheets and claims:
| Fmoc | 9-fluorenylmethyloxycarbonyl |
| Boc | Tertbutyloxycarbonyl |
| tBu | The tertiary butyl |
| Trt | Trityl |
| NMP | N-Methyl pyrrolidone |
| DMSO | DMSO 99.8MIN. |
| DMF | N, dinethylformamide |
| DCM | Methylene dichloride |
| DBLK | 20% hexahydropyridine/DMF solution |
| DIC | N, the N-DIC |
| DIPEA | N, the N-diisopropylethylamine |
| DMAP | The 4-dimethylamino pyridine |
| PYBOP | Phosphofluoric acid benzotriazole-1-base-oxygen base tripyrrole alkyl |
| TBTU | O-benzotriazole-N, N, N', N'-tetramethyl-urea Tetrafluoroboric acid |
| HOBT | I-hydroxybenzotriazole |
| TFE | Trifluoroethanol |
| TFA | Trifluoroacetic acid |
| EDT | 1 |
| PHOH | Phenol |
| TIS | Tri isopropyl silane |
Embodiment 1: according to strategy 1 preparation teriparatide
One, peptide resin fragment 1 (teriparatide sequence 34-26 amino acids)
1.Fmoc-Phe-Wang Resin preparation
Taking by weighing substitution degree is the Wang Resin 1.2g of 0.8mmol/g, joins in the solid state reaction post, with DMF washing 2 times; With DMF swelling resin after 30 minutes; Take by weighing 0.51g Fmoc-Phe-OH, 0.13gHOBT and 0.01gDMAP and dissolve, after ice-water bath adds down 0.16mL DIC activation, add in the above-mentioned reaction column that resin is housed with DMF; React after 2 hours, add 5mL pyridine and 5.5ml diacetyl oxide sealing 12 hours.With DMF washing 6 times, obtain Fmoc-Phe – Wang Resin, the detection substitution degree is 0.20mmol/g.
2. the segmental preparation of peptide resin
Taking by weighing substitution degree is the Fmoc-Phe-Wang Resin 1.5g of 0.20mmol/g, adds in the solid state reaction post, with DMF washing 2 times, after 30 minutes, removes Fmoc protection with DBLK with DMF swelling Fmoc-Phe-Wang Resin, then with DMF washing 6 times.With 0.90g Fmoc-Asn (Trt)-OH, 0.25g HOBt, 0.29ml DIC are dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1; Add in the solid state reaction post, (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if the resin water white transparency; Then react completely; The resin colour developing, the expression reaction not exclusively needs linked reaction 1h again).Repeat the above-mentioned Fmoc of removing protection and add corresponding amino acid link coupled step; Order according to peptide resin fragment 1 (sequence (34-26)); Accomplish Fmoc-His (Trt)-OH, Fmoc-Val-OH, Fmoc-Asp (OtBu)-OH, Fmoc-Gln (Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc)-OH, Fmoc-Lys (Boc)-OH successively; Remove the Fmoc protection with DBLK at last, obtain peptide resin fragment 1.
Two, the preparation of side chain protected peptide fragment 2 (teriparatide sequence 25-21 amino acids)
1. the preparation of peptide fragment 2 (teriparatide sequence 25-21 amino acids) peptide resin
Taking by weighing substitution degree is the 2-CTC resin 5.26g of 0.47mmol/g, joins in the solid state reaction post, with DMF washing 2 times;, get 3.24g Fmoc-Arg (pbf)-OH and dissolve after 30 minutes with DMF swelling resin, after ice-water bath adds 1.7mL DIPEA activation down with DMF; Add in the above-mentioned reaction column that resin is housed; React after 2 hours, add 10mL anhydrous methanol sealing 1 hour, with DMF washing 6 times.Remove the Fmoc protection with DBLK, then with DMF washing 6 times.
With 1.77g Fmoc-Leu-OH, 0.74g HOBt, 0.86ml DIC are dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1; Add in the solid state reaction post, (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if the resin water white transparency; Then react completely; The resin colour developing, the expression reaction not exclusively needs linked reaction 1h again).Repeat the above-mentioned Fmoc of removing protection and add corresponding amino acid link coupled step; Order according to peptide fragment 2 (sequence (25-21)); Accomplish Fmoc-Trp (Boc)-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Val-OH successively, wherein last amino acid Val coupling need not remove Fmoc after finishing.Reaction finishes the back shrinks with methyl alcohol, the resin drying under vacuum overnight, and weighing obtains peptide fragment 2 (teriparatide sequence 25-21 amino acids) peptide resin 8.12g.
2. cleavage of peptide fragment 2 (teriparatide sequence 25-21 amino acids) peptide resin, side chain protected peptide fragment 2 (teriparatide sequence 25-21 amino acids)
Take by weighing peptide fragment 2 (teriparatide sequence 25-21 amino acids) peptide resin 8.12g, be added in the 250ml flask.(volume ratio THE:DCM=1:4), is poured lytic reagent in the flask into room temperature reaction 2h to configuration lytic reagent 82ml.Reaction finishes, and filters resin, collects filtrating.Filtrate volume revolved steam to ﹤ 25vol%, drop in the 200ml precipitation reagent (volume ratio, normal hexane: ether=1:4); Centrifugal, anhydrous diethyl ether washing, and vacuum-drying; Obtain side chain protected peptide fragment 2 (teriparatide sequence 25-21 amino acids); With the water-soluble back freeze-drying of bullion, get 0.7g side chain protected peptide fragment 2 (teriparatide sequence 25-21 amino acids), purity 95%.
Prepare side chain protected peptide fragment 3, side chain protected peptide fragment 4, side chain protected peptide fragment 5 as stated above.
Three, the preparation of peptide resin I
Take by weighing 0.7g side chain protected peptide fragment 2 (teriparatide sequence 25-21 amino acids), 1.9g TBTU, 0.74g HOBt, use DMF and DMSO to add 1.7mlDIPEA as the mixed solution dissolving back of 1:1, mixed solution adds in the step 1 solid state reaction post; (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 3h; If the resin water white transparency then reacts completely, the resin colour developing; The expression reaction not exclusively needs linked reaction 1h again).Remove the Fmoc protection with DBLK, with DMF washing 6 times, obtain the peptide resin I then.
Four, the preparation of peptide resin II
Take by weighing 0.9g side chain protected peptide fragment 3,2.6gPyBOP, 0.74gHOBt, use DMF and NMP to add 1.7mlDIPEA as the mixed solution of 1:1 dissolving back, mixed solution adds in the step 3 in the peptide resin solid state reaction post; (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 3h; If the resin water white transparency then reacts completely, the resin colour developing; The expression reaction not exclusively needs linked reaction 1h again).Remove the Fmoc protection with DBLK, with DMF washing 6 times, obtain the peptide resin II then.
Five, the preparation of peptide resin III
Take by weighing 0.55g side chain protected peptide fragment 4,2.6gPyBOP, 0.74gHOBt, use DMF and DMSO to add 1.7mlDIPEA as the mixed solution of 1:1 dissolving back, mixed solution adds in the step 4 in the peptide resin solid state reaction post; (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 3h; If the resin water white transparency then reacts completely, the resin colour developing; The expression reaction not exclusively needs linked reaction 1h again).Remove the Fmoc protection with DBLK, with DMF washing 6 times, obtain the peptide resin III then.
Six, the preparation of peptide resin IV
Take by weighing 0.82g side chain protected peptide fragment 5,2.6gPyBOP, 0.74gHOBt, use DMF and DMSO to add 1.7ml DIPEA as the mixed solution of 1:1 dissolving back, mixed solution adds in the step 5 in the peptide resin solid state reaction post; (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 3h; If the resin water white transparency then reacts completely, the resin colour developing; The expression reaction not exclusively needs linked reaction 1h again).Remove the Fmoc protection with DBLK, then with DMF washing 6 times, reaction finishes the back shrinks with methyl alcohol, the resin drying under vacuum overnight, and weighing obtains 3.1g peptide resin IV.
Seven, the cracking of peptide resin IV obtains the bullion teriparatide
3.1g peptide resin IV in the step 6 is joined in the 100ml flask, and the configuration lytic reagent (TFA:DET:TIS:PHOH:H2O=80:5:5:5:5), pour lytic reagent in the flask into, room temperature reaction 3 hours by volume ratio.Reaction finishes, and filters resin, collects filtrating.Drop in the 300ml ether reagent, centrifugal, anhydrous diethyl ether washing, and vacuum-drying obtain 1.2g. teriparatide bullion, purity 75.4%.
Eight, the purified teriparatide that obtains of teriparatide bullion
Take by weighing the thick peptide of 1.2g teriparatide with the 50mL water dissolution after, adopt Waters 2695RP-HPLC system, wavelength 230nm; Chromatographic column is 50 * 250mm anti-phase C18 post; Rule 0.2%TFA/ acetonitrile moving phase purifying is collected purpose peak cut, obtains purity greater than 98.5% smart peptide.Smart peptide solution is adopted Waters 2695RP-HPLC system; Chromatographic column is 50 * 250mm anti-phase C18 post, and 0.2% acetum/acetonitrile moving phase is changeed salt, collects purpose peak cut; Rotary evaporation concentrates; Freeze-drying obtains the smart peptide 0.4g of teriparatide acetate salt, HPLC purity 99.3%, total recovery 32.2%.
Embodiment 2: according to strategy 2 preparation teriparatides
One, the preparation of side chain protected peptide fragment b (teriparatide sequence 25-11 amino acids)
1. the preparation of side chain protected peptide fragment b peptide resin
Taking by weighing substitution degree is the 2-CTC resin 5.35g of 0.47mmol/g, joins in the solid state reaction post, with DMF washing 2 times;, get 3.24g Fmoc-Arg (pbf)-OH and dissolve after 30 minutes with DMF swelling resin, after ice-water bath adds 1.7mL DIPEA activation down with DMF; Add in the above-mentioned reaction column that resin is housed; React after 2 hours, add 10mL anhydrous methanol sealing 1 hour, with DMF washing 6 times.Remove the Fmoc protection with DBLK, then with DMF washing 6 times.
With 1.77g Fmoc-Leu-OH, 0.74g HOBt, 0.86ml DIC are dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1; Add in the solid state reaction post, (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if the resin water white transparency; Then react completely; The resin colour developing, the expression reaction not exclusively needs linked reaction 1h again).Repeat the above-mentioned Fmoc of removing protection and add corresponding amino acid link coupled step,, accomplish Fmoc-Trp (Boc)-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Val-OH successively according to the order of peptide fragment b (teriparatide sequence 25-11 amino acids); Fmoc-Arg (Pbf)-OH; Fmoc-Glu (OtBu)-OH, Fmoc-Met-OH, Fmoc-Ser (tBu)-OH Fmoc-Asn (Trt)-OH; Fmoc-Leu-OH; Fmoc-His (Trt)-OH, Fmoc-Lys (Boc)-OH Fmoc-Gly-OH, Fmoc-Leu-OH.Wherein last amino acid Leu coupling need not remove Fmoc after finishing.Reaction finishes the back shrinks with methyl alcohol, the resin drying under vacuum overnight, and weighing obtains side chain protected peptide fragment b peptide resin 15.4g.
2. cracking side chain protected peptide fragment b peptide resin, side chain protected peptide fragment b
Side chain protected peptide fragment b peptide resin 15.4g is added in the 250ml flask.(volume ratio THE:DCM=1:4), is poured lytic reagent in the flask into room temperature reaction 2h to configuration lytic reagent 150ml.Reaction finishes, and filters resin, collects filtrating.Filtrate volume revolved steam to ﹤ 25vol%, drop in the 200ml precipitation reagent (volume ratio, normal hexane: ether=1:4); Centrifugal, anhydrous diethyl ether washing, and vacuum-drying; Obtain side chain protected peptide fragment b; With the water-soluble back freeze-drying of bullion, get 1.4g side chain protected peptide fragment sequence (25-11), purity 85%.
Two, the preparation of side chain protected peptide fragment c
1. the preparation of side chain protected peptide fragment c peptide resin
Taking by weighing substitution degree is the 2-CTC resin 5.33g of 0.47mmol/g, joins in the solid state reaction post, with DMF washing 2 times;, get 3.11g Fmoc-Asn (Trt)-OH and dissolve after 30 minutes with DMF swelling resin, after ice-water bath adds 1.7mL DIPEA activation down with DMF; Add in the above-mentioned reaction column that resin is housed; React after 2 hours, add 10mL anhydrous methanol sealing 1 hour, with DMF washing 6 times.Remove the Fmoc protection with DBLK, then with DMF washing 6 times.
With 3.25g Fmoc-His (Trt)-OH, 0.74g HOBt, 0.86ml DIC are dissolved in DCM and the DMF mixing solutions that volume ratio is 1:1; Add in the solid state reaction post, (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 2h, if the resin water white transparency; Then react completely; The resin colour developing, the expression reaction not exclusively needs linked reaction 1h again).Repeat the above-mentioned Fmoc of removing protection and add corresponding amino acid link coupled step,, accomplish Fmoc-Met-OH successively according to the order of side chain protected peptide fragment c; Fmoc-Leu-OH; Fmoc-Gln (Trt)-OH, Fmoc-IIe-OH, Fmoc-Glu (OtBu)-OH; Fmoc-Ser (tBu)-OH, Fmoc-Val-OH Fmoc-Ser (tBu)-OH.Wherein last amino acid Ser coupling need not remove Fmoc after finishing.Reaction finishes the back shrinks with methyl alcohol, the resin drying under vacuum overnight, and weighing obtains peptide fragment sequence (10-1) peptide resin 14.6g.
2. cracking side chain protected peptide fragment c peptide resin, side chain protected peptide fragment c
Side chain protected peptide fragment c peptide resin 14.6g is added in the 250ml flask.(volume ratio THE:DCM=1:4), is poured lytic reagent in the flask into room temperature reaction 2h to configuration lytic reagent 150ml.Reaction finishes, and filters resin, collects filtrating.Filtrate volume revolved steam to ﹤ 25vol%, drop in the 200ml precipitation reagent (volume ratio, normal hexane: ether=1:4); Centrifugal, anhydrous diethyl ether washing, and vacuum-drying; Obtain side chain protected peptide fragment c; With the water-soluble back freeze-drying of bullion, get 1.3g side chain protected peptide fragment sequence (10-1), purity 80.5%.
Three, side chain protected peptide fragment b and embodiment 1 peptide resin fragment 1 (teriparatide sequence 34-26 amino acids) condensation reaction gets peptide resin A
Take by weighing 1.4g side chain protected peptide fragment b, 1.9g TBTU, 0.74g HOBt, use DMF and DMSO are that 1: 1 mixed solution dissolving back adds 1.7mlDIPEA, and mixed solution adds in the peptide resin fragment 1 solid state reaction post that obtains among the embodiment 1; (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 3h; If the resin water white transparency then reacts completely, the resin colour developing; The expression reaction not exclusively needs linked reaction 1h again).Remove the Fmoc protection with DBLK, with DMF washing 6 times, obtain peptide resin A then.
Four, the condensation reaction of side chain protected peptide fragment c and peptide resin A gets peptide resin B
Get 1.3g side chain protected peptide fragment c, 1.9g TBTU, 0.74g HOBt, use DMF and DMSO to add 1.7mlDIPEA as the mixed solution dissolving back of 1:1, mixed solution adds in the step 3 solid state reaction post; (reaction end detects with ninhydrin method and is as the criterion room temperature reaction 3h; If the resin water white transparency then reacts completely, the resin colour developing; The expression reaction not exclusively needs linked reaction 1h again.Remove the Fmoc protection with DBLK, with DMF washing 6 times, obtain 2.8g peptide resin B then.
Five, peptide resin B cracking obtains the bullion teriparatide
2.8g peptide resin B in the step 4 is joined in the 100ml flask configuration lytic reagent (volume ratio, TFA:DET:TIS:PHOH:H
2O=80:5:5:5:5), lytic reagent is poured in the flask into room temperature reaction 3 hours.Reaction finishes, and filters resin, collects filtrating.Drop in the 300ml ether reagent, centrifugal, anhydrous diethyl ether washing, and vacuum-drying obtain 1.0g. teriparatide bullion, purity 80.5%.
Six, the purified teriparatide that obtains of teriparatide bullion
Take by weighing the thick peptide of 1.0g teriparatide with the 50mL water dissolution after, adopt Waters 2695RP-HPLC system, wavelength 230nm; Chromatographic column is 50 * 250mm anti-phase C18 post; Rule 0.2%TFA/ acetonitrile moving phase purifying is collected purpose peak cut, obtains purity greater than 98.5% smart peptide.Smart peptide solution is adopted Waters 2695RP-HPLC system; Chromatographic column is 50 * 250mm anti-phase C18 post, and 0.2% acetum/acetonitrile moving phase is changeed salt, collects purpose peak cut; Rotary evaporation concentrates; Freeze-drying obtains the smart peptide 0.25g of teriparatide acetate salt, HPLC purity 99.1%, total recovery 20.5%.
Embodiment 3:
Respectively according to strategy 3,4 and 5 preparation teriparatides.The thick peptide of teriparatide that statistics each side method makes and the purity and the total recovery of the smart peptide of teriparatide acetate, the result sees table 1.
The teriparatide result that each side's method makes among table 1 embodiment 1 ~ 3
The explanation of above embodiment just is used for helping to understand method of the present invention and core concept thereof.Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention, can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of claim of the present invention.
Claims (15)
1. the preparation method of a teriparatide; It is characterized in that each peptide resin fragment of teriparatide is formed in preparation, with each peptide resin fragment on solid phase progressively coupling obtain the teriparatide resin; Cracking obtains the teriparatide bullion then, and purifying obtains the product teriparatide.
2. according to the said preparation method of claim 1, it is characterized in that said preparation method specifically may further comprise the steps:
A) 5 peptide fragment of teriparatide are formed in preparation: peptide resin fragment 1, side chain protected peptide fragment 2; Side chain protected peptide fragment 3, side chain protected peptide fragment 4 and side chain protected peptide fragment 5; Wherein said peptide resin fragment 1 is a teriparatide sequence 34-26 amino acids; Said side chain protected peptide fragment 2 is a teriparatide sequence 25-21 amino acids; Said side chain protected peptide fragment 3 is a teriparatide sequence 20-13 amino acids, and said side chain protected peptide fragment 4 is a teriparatide sequence 12-7 amino acids, and said side chain protected peptide fragment 5 is a teriparatide sequence 6-1 amino acids;
B) side chain protected peptide fragment 2 and 1 coupling of peptide resin fragment are obtained the peptide resin I;
C) side chain protected peptide fragment 3 and the coupling of peptide resin I are obtained the peptide resin II;
D) side chain protected peptide fragment 4 and the coupling of peptide resin II are obtained the peptide resin III;
E) side chain protected peptide fragment 5 and the coupling of peptide resin III are obtained the peptide resin IV;
F) cracking of peptide resin IV obtains the teriparatide bullion
G) the purified teriparatide that obtains of teriparatide bullion.
3. according to the said preparation method of claim 2, it is characterized in that said steps A) in peptide resin fragment 1 used solid phase carrier be king's resin.
4. according to the said preparation method of claim 2; It is characterized in that; Said steps A) side chain protected peptide fragment 2, side chain protected peptide fragment 3, side chain protected peptide fragment 4 and side chain protected peptide fragment 5 are coupled at resin by each segmental amino acid respectively and obtain each segmental peptide resin in, and cracking makes then.
5. according to the said preparation method of claim 4, it is characterized in that said steps A) in side chain protected peptide fragment 2, side chain protected peptide fragment 3, side chain protected peptide fragment 4 and side chain protected peptide fragment 5 used solid phase carriers be 2-chlorine trityl chloride resin.
6. according to the said preparation method of claim 4; It is characterized in that said steps A) in the coupling agent of peptide resin fragment 1, side chain protected peptide fragment 2, side chain protected peptide fragment 3, side chain protected peptide fragment 4 and side chain protected peptide fragment 5 be HOBT/DIC, PyBOP/HOBt/DIPEA or TBTU/HOBt/DIPEA.
7. according to the said preparation method of claim 4, it is characterized in that said steps A) in the cracking agent of side chain protected peptide fragment 2, side chain protected peptide fragment 3, side chain protected peptide fragment 4, side chain protected peptide fragment 5 be TFE.
8. according to the said preparation method of claim 2, it is characterized in that said step B)-step e) in side chain protected peptide and peptide resin link coupled coupling agent be HOBT/DIC, PyBOP/HOBt/DIPEA or TBTU/HOBt/DIPEA.
9. according to the said preparation method of claim 2, it is characterized in that said step B)-step e) in side chain protected peptide fragment and peptide resin link coupled reaction solvent be among DMF, DCM, NMP, the DMSO one or more.
10. according to the said preparation method of claim 2; It is characterized in that said step F) in peptide resin IV cracked lysate be that TFA and water volume ratio are that mixing solutions, TFA, DET, PHOH and the water volume ratio of 95:5 is that mixing solutions or TFA, DET, TIS, PHOH and the water volume ratio of 90:5:3:2 is the mixing solutions of 80:5:5:5:5.
11., it is characterized in that said step G according to the said preparation method of claim 2) in purifying be the RPLC purifying.
12., it is characterized in that said preparation method specifically may further comprise the steps according to the said preparation method of claim 1:
1) 3 peptide fragment of teriparatide are formed in preparation: peptide resin fragment a, side chain protected peptide fragment b and side chain protected peptide fragment c; Wherein said peptide resin fragment a is a teriparatide sequence 34-26 amino acids; Said side chain protected peptide fragment b is a teriparatide sequence 25-11 amino acids, and said side chain protected peptide fragment c is a teriparatide sequence 10-1 amino acids;
2) side chain protected peptide fragment b and peptide resin fragment a coupling are obtained peptide resin A;
3) side chain protected peptide fragment 3 and peptide resin A coupling are obtained peptide resin B;
4) peptide resin B cracking obtains the teriparatide bullion;
5) the purified teriparatide that obtains of teriparatide bullion.
13., it is characterized in that said preparation method specifically may further comprise the steps according to the said preparation method of claim 1:
A) 4 peptide fragment of teriparatide are formed in preparation: peptide resin Segment A, side chain protected peptide fragment B; Side chain protected peptide fragment C and side chain protected peptide fragment D; Wherein said peptide resin Segment A is a teriparatide sequence 34-29 amino acids; Said side chain protected peptide fragment B is a teriparatide sequence 28-16 amino acids, and said side chain protected peptide fragment C is a teriparatide sequence 15-7 amino acids, and said side chain protected peptide fragment D is a teriparatide sequence 6-1 amino acids;
B) side chain protected peptide fragment B and the coupling of peptide resin Segment A are obtained peptide resin i;
C) side chain protected peptide fragment C and peptide resin i coupling are obtained peptide resin ii;
D) side chain protected peptide fragment D and peptide resin ii coupling are obtained peptide resin iii;
E) peptide resin iii cracking obtains the teriparatide bullion;
F) the purified teriparatide that obtains of teriparatide bullion.
14., it is characterized in that said preparation method specifically may further comprise the steps according to the said preparation method of claim 1:
I) 5 peptide fragment of teriparatide are formed in preparation: the peptide resin fragment 1., the side chain protected peptide fragment 2.; The side chain protected peptide fragment 3., the side chain protected peptide fragment 4. with the side chain protected peptide fragment 5.; 1. wherein said peptide resin fragment is teriparatide sequence 34-29 amino acids; 2. said side chain protected peptide fragment is teriparatide sequence 28-20 amino acids; 3. said side chain protected peptide fragment is teriparatide sequence 19-13 amino acids, and 4. said side chain protected peptide fragment is teriparatide sequence 12-7 amino acids, and 5. said side chain protected peptide fragment is teriparatide sequence 6-1 amino acids;
II) side chain protected peptide fragment 2 and 1 coupling of peptide resin fragment are obtained peptide resin a;
III) side chain protected peptide fragment 3 and peptide resin a coupling are obtained peptide resin b;
IV) side chain protected peptide fragment 4 and peptide resin b coupling are obtained peptide resin c;
V) side chain protected peptide fragment 5 and peptide resin c coupling are obtained peptide resin d;
VI) peptide resin d cracking obtains the teriparatide bullion;
VII) the purified teriparatide that obtains of teriparatide bullion.
15., it is characterized in that said preparation method specifically may further comprise the steps according to the said preparation method of claim 1:
I) 5 peptide fragment of teriparatide are formed in preparation: peptide resin fragment I, side chain protected peptide fragment II; Side chain protected peptide fragment III, side chain protected peptide fragment IV and side chain protected peptide fragment V; Wherein said peptide resin fragment I is a teriparatide sequence 34-29 amino acids; Said side chain protected peptide fragment II is a teriparatide sequence 28-21 amino acids; Said side chain protected peptide fragment III is a teriparatide sequence 20-13 amino acids, and said side chain protected peptide fragment IV is a teriparatide sequence 12-7 amino acids, and said side chain protected peptide fragment V is a teriparatide sequence 6-1 amino acids;
Ii) 1. side chain protected peptide fragment II and the coupling of peptide resin fragment I are obtained peptide resin;
Iii) with side chain protected peptide fragment III and peptide resin 1. coupling obtain peptide resin 2.;
Iv) with side chain protected peptide fragment IV and peptide resin 2. coupling obtain peptide resin 3.;
V) with side chain protected peptide fragment V and peptide resin 3. coupling obtain peptide resin 4.;
Vi) peptide resin 4. cracking obtain the teriparatide bullion;
The vii) purified teriparatide that obtains of teriparatide bullion.
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| CN102993293A (en) * | 2012-12-05 | 2013-03-27 | 深圳翰宇药业股份有限公司 | Method for purifying teriparatide acetate |
| CN103467595A (en) * | 2013-09-06 | 2013-12-25 | 深圳翰宇药业股份有限公司 | Method for preparing teriparatide |
| CN104017064A (en) * | 2014-06-13 | 2014-09-03 | 杭州诺泰制药技术有限公司 | Method for preparing teriparatide |
| CN104277097A (en) * | 2014-10-22 | 2015-01-14 | 申联生物医药（上海）有限公司 | Method for preparing synthetic peptide antigen 2700 of swine O-type foot and mouth disease through solid-phase fragment process |
| CN104277098A (en) * | 2014-10-22 | 2015-01-14 | 申联生物医药（上海）有限公司 | Method for preparing synthetic peptide antigen 2600 of swine O-type foot and mouth disease through solid-phase fragment process |
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| CN109096388A (en) * | 2017-07-24 | 2018-12-28 | 江苏金斯瑞生物科技有限公司 | A kind of preparation method of Teriparatide |
| WO2020000555A1 (en) * | 2018-06-26 | 2020-01-02 | 深圳翰宇药业股份有限公司 | Method for preparing teriparatide |
| CN111057139A (en) * | 2018-10-17 | 2020-04-24 | 南京华威医药科技集团有限公司 | Novel process for preparing teriparatide |
| CN111848778A (en) * | 2019-04-30 | 2020-10-30 | 上海医药工业研究院 | Teriparatide analogues |
| CN112625117A (en) * | 2020-12-23 | 2021-04-09 | 无锡和邦生物科技有限公司 | Non-denaturing purification method and application of soluble recombinant teriparatide |
| CN114137114A (en) * | 2020-11-26 | 2022-03-04 | 信立泰(苏州)药业有限公司 | Teriparatide isotope internal standard, preparation method and application thereof |
| 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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| CN111057139B (en) * | 2018-10-17 | 2023-12-22 | 南京华威医药科技集团有限公司 | Novel process for preparing teriparatide |
| CN111848778A (en) * | 2019-04-30 | 2020-10-30 | 上海医药工业研究院 | Teriparatide analogues |
| CN111848778B (en) * | 2019-04-30 | 2024-03-19 | 上海医药工业研究院 | Teriparatide analogues |
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| 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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Application publication date: 20121017 |