CN105777872B - A kind of purification process of Sa Molu peptide - Google Patents
A kind of purification process of Sa Molu peptide Download PDFInfo
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- CN105777872B CN105777872B CN201410784130.9A CN201410784130A CN105777872B CN 105777872 B CN105777872 B CN 105777872B CN 201410784130 A CN201410784130 A CN 201410784130A CN 105777872 B CN105777872 B CN 105777872B
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- 238000000746 purification Methods 0.000 title claims abstract description 100
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- 238000004587 chromatography analysis Methods 0.000 description 1
- 208000035850 clinical syndrome Diseases 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 208000016097 disease of metabolism Diseases 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000035619 diuresis Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
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- 238000009472 formulation Methods 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 238000006317 isomerization reaction Methods 0.000 description 1
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 1
- 208000030159 metabolic disease Diseases 0.000 description 1
- 150000002825 nitriles Chemical class 0.000 description 1
- 229940038384 octadecane Drugs 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- NFHFRUOZVGFOOS-UHFFFAOYSA-N palladium;triphenylphosphane Chemical compound [Pd].C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1.C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 NFHFRUOZVGFOOS-UHFFFAOYSA-N 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- PARWUHTVGZSQPD-UHFFFAOYSA-N phenylsilane Chemical compound [SiH3]C1=CC=CC=C1 PARWUHTVGZSQPD-UHFFFAOYSA-N 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 229910000028 potassium bicarbonate Inorganic materials 0.000 description 1
- 235000015497 potassium bicarbonate Nutrition 0.000 description 1
- 239000011736 potassium bicarbonate Substances 0.000 description 1
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- GCYXWQUSHADNBF-AAEALURTSA-N preproglucagon 78-108 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 GCYXWQUSHADNBF-AAEALURTSA-N 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000012521 purified sample Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 108010060325 semaglutide Proteins 0.000 description 1
- 229950011186 semaglutide Drugs 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
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- 239000002904 solvent Substances 0.000 description 1
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Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention belongs to field of pharmaceutical chemistry technology, disclose a kind of purification process of Sa Molu peptide.The purification process includes: to take Sa Molu peptide sample to be purified, is separated through chemical bonding silica gel chromatographic column first, carries out gradient elution with A1 solution and the mixed solution of B solution, collects elution fraction, obtain first time purified；First time purified is taken, is separated through chemical bonding silica gel chromatographic column second, gradient elution is carried out with A2 solution and the mixed solution of B solution, collects elution fraction, get Sa Molu peptide solution；A1 solution is carbonate solution；A2 solution is phosphate solution；B solution is the mixed solution of acetonitrile and isopropanol, and the ratio between acetonitrile and the volume of isopropanol are (8:2)~(9:1).The Sa Molu peptide purity is high that the purification process prepares, significantly improves the yield of Sa Molu peptide, and easy to operate, is advantageously implemented the prepare with scale of Sa Molu peptide.
Description
Technical field
The invention belongs to field of pharmaceutical chemistry technology, in particular to a kind of purification process of Sa Molu peptide.
Background technique
Diabetes are a series of a kind of clinical syndromes because caused by internal insulin is absolute or relative deficiency, with something lost
Passing gene has very close association.The main clinical manifestation of diabetes is more drinks, diuresis, eat and weight loss more, and
Contain glucose etc. in blood glucose height, urine.Diabetes are divided into four seed types: Type I diabetes, type II diabetes, other types sugar
Urine disease and gestational diabetes.Any kind of diabetes can all cause the β cell in pancreas that cannot generate enough insulin
To reduce the concentration of blood glucose, lead to the generation of hyperglycemia.World Health Organization's report in 2011 points out that the whole world has 3.46
Hundred million people suffer from diabetes, are estimated to be 3,400,000 people within 2004 and die of disease caused by hyperglycemia, the Diabetes Death hair more than 80%
Life is in low income and middle income country.
Type II diabetes is once called as Non-Insulin Dependent Diabetes Mellitus (NIDDM) or adult onset diabetes (adult-
Onset diabetes), it is a kind of metabolic disease, it is characterized in that hyperglycemia, mainly opposite by insulin resistance and insulin
Shortage causes.Wherein, patients with NIDDM accounts for 90% or so in diabetic, remaining 10% predominantly Type I diabetes
With gestational diabetes, so the research and development of drug for type II diabetes have a very important significance.
Many for the antidiabetic drug type of type II diabetes, the receptor agonism element of glucagon-like peptide-1 (GLP-1) is close
The hot spot studied over year.Wherein, Sa Molu peptide (semaglutide) is one of receptor agonism element of GLP-1, which is by pellet
What wheat Novo Nordisk Co., Ltd developed.Sa Molu peptide can play good as a kind of subcutaneous injection formulation expendable weekly
The effect of blood glucose is reduced, while also having effects that weight-reducing, chemistry is expressed as Nε26-{18-[N-(17-
carboxyheptadecanoyl)-L-γ-glutamyl]-10-oxo-3,6,12,15-tetraoxa-9,18-
diazaoctade canoyl}-[8-(2-amino-2-propanoic acid),34-L-arginine]human
Glucagon-like peptide 1 (7-37), with structure shown in Formulas I:
Currently, the preparation method of Sa Molu peptide is mainly chemical synthesis.Since the peptide chain of Sa Molu peptide is longer, side chain contains
Have longer modification, in peptide sequence containing in such as Ser synthesis process easily the amino acid of isomerization and cause in thick peptide with isomery
Body impurity needs to carry out the thick peptide of Sa Molu peptide obtained by chemical synthesis further so the impurity generated in synthesis is more
Purifying.However, the yield of Sa Molu peptide purification method is lower in the prior art, the purifying of Sa Molu peptide is caused to become Sa Molu
One of difficult point, the bottleneck of Sa Molu peptide industrialization in peptide preparation process.It is therefore desirable to explore a kind of purifying side of Sa Molu peptide
Method, to improve the yield of Sa Molu peptide.
Summary of the invention
In view of this, goal of the invention of the invention is to provide a kind of purification process of Sa Molu peptide.It is provided by the invention
The purification process of Sa Molu peptide is easy to operate, improves the yield of Sa Molu peptide, and the industrialization for being more advantageous to Sa Molu peptide is raw greatly
It produces.
In order to realize that goal of the invention of the invention, the present invention adopt the following technical scheme that:
The present invention provides a kind of purification process of Sa Molu peptide comprising:
Sa Molu peptide sample to be purified is taken, is separated through chemical bonding silica gel chromatographic column first, with A1 solution and B solution
Mixed solution carries out gradient elution, collects elution fraction, obtains first time purified；
Gained first time purified is taken, is separated through chemical bonding silica gel chromatographic column second, with the mixing of A2 solution and B solution
Solution carries out gradient elution, collects elution fraction, get Sa Molu peptide solution；
A1 solution is carbonate solution；
A2 solution is phosphate solution；
B solution be acetonitrile and isopropanol mixed solution, wherein the ratio between volume of acetonitrile and isopropanol be (8:2)~(9:
1)。
The peptide chain of Sa Molu peptide is longer, branched structure is complicated, and the impurity generated in the synthesis process is more, so needing pairing
At Sa Molu peptide purified.In the present invention, by research discovery due in the peptide sequence of Sa Molu peptide contain hydrophobicity compared with
Strong amino acid, it is lower to cause the solubility of Sa Molu peptide in aqueous solution, and difficult lower prop, causes damages in purification process, increases
Add purifying difficulty, affects the yield of Sa Molu peptide.The present invention is by be chemically bonded silica gel as stationary phase, after loading,
Gradient elution is carried out with carbonate, most of isomer impurities in Sa Molu peptide to be purified can be difficult to separate with other
Impurity separation, removal；Gradient elution is carried out as mobile phase using phosphate solution later, removal is difficult to remove minute impurities simultaneously
Efficiently solve the problems, such as that difficult lower prop, yield are low.Experimental result confirms that Sa Molu peptide obtained by purification process provided by the invention is pure
Degree height, high income, and it is easy to operate, it is advantageously implemented the prepare with scale of Sa Molu peptide.
In some embodiments of the invention, in purification process provided by the invention, the body of acetonitrile and isopropanol in B solution
The ratio between product is 8:2 or 9:1.
Preferably, in purification process provided by the invention, the concentration of carbonate solution used in the first separation process is
20mmol/L~150mmol/L.In some embodiments of the invention, in purification process provided by the invention, carbonate solution
Concentration be 20mmol/L, 50mmol/L, 100mmol/L or 150mmol/L.
Preferably, in purification process provided by the invention, in the first separation process the pH value of A1 solution used be pH7.5~
pH8.5.In some embodiments of the invention, in purification process provided by the invention, the pH value of A1 solution is pH7.5, pH8.0
Or pH8.5.In other embodiment of the invention, in purification process provided by the invention, with tetramethylammonium hydroxide tune
The pH value of A1 solution is saved to pH7.5~pH8.5.
Preferably, in purification process provided by the invention, the concentration of phosphate solution used in the second separation process is
20mmol/L~150mmol/L.In other embodiment of the invention, in purification process provided by the invention, phosphate
The concentration of buffer is 20mmol/L, 50mmol/L, 100mmol/L or 150mmol/L.
Preferably, in purification process provided by the invention, the pH value of A2 solution used in the second separation process is pH2.0
~pH3.0.In some embodiments of the invention, in purification process provided by the invention, the pH value of A2 solution be pH2.0,
PH2.5 or pH3.0.In other embodiment of the invention, in purification process provided by the invention, it is molten that A2 is adjusted with phosphoric acid
The pH to pH2.0~pH3.0 of liquid.
Preferably, in purification process provided by the invention, in the first separation process, A1 is molten in mobile phase used in gradient elution
Liquid and the Volume fraction of B solution are 62%:38% → 50%:50%.
Preferably, in purification process provided by the invention, in the second separation process, in mobile phase used in gradient elution, A2
Solution and the Volume fraction of B solution are 60%:40% → 50%:50%.
Preferably, the carbon in purification process provided by the invention, in the first separation process, in mobile phase used in gradient elution
Hydrochlorate is the above mixture of one or both of ammonium carbonate, ammonium hydrogen carbonate or saleratus.
Preferably, the phosphorus in purification process provided by the invention, in the second separation process, in mobile phase used in gradient elution
Acid salt solution, which is that one or both of sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate are above, to be mixed
Object.
Preferably, in purification process provided by the invention, in the first separation process, flowing corresponding to elution fraction is collected
A1 solution and the Volume fraction of B solution are 56%:44% → 52%:48% in phase.
Preferably, in purification process provided by the invention, in the second separation process, flowing corresponding to elution fraction is collected
A2 solution and the Volume fraction of B solution are 54%:46% → 51%:49% in phase.
Preferably, in purification process provided by the invention, silica gel chromatographic column is chemically bonded used in the first separation process
For C8 silica gel chromatographic column or butyl bonded silica gel chromatographic column.In some embodiments of the invention, purifying side provided by the invention
In method, chemical bonding silica gel chromatographic column used in the first separation process is butyl bonded silica gel chromatographic column.
Preferably, in purification process provided by the invention, silica gel chromatographic column is chemically bonded used in the second separation process
For C8 silica gel chromatographic column or butyl bonded silica gel chromatographic column.In some embodiments of the invention, purifying side provided by the invention
In method, chemical bonding silica gel chromatographic column used in the second separation process is butyl bonded silica gel chromatographic column.
In the present invention, in purification process provided by the invention, silica gel color is chemically bonded used in the first separation process
Chemical bonding silica gel chromatographic column used in spectrum column, the second separation process may be the same or different, and the two is independent of each other.
In some embodiments of the invention, in purification process provided by the invention, silicon is chemically bonded used in the first separation process
Chemical bonding silica gel used in glue chromatographic column, the second separation process is identical.
In some embodiments of the invention, in purification process provided by the invention, change used in the first separation process
Learn bonded silica gel chromatographic column specification include:
5cm × 25cm (pillar diameter × length), 10cm × 25cm (pillar diameter × length), 15cm × 25cm (pillar
Diameter × length).
In some embodiments of the invention, in purification process provided by the invention, change used in the second separation process
Learn bonded silica gel chromatographic column specification include:
5cm × 25cm (pillar diameter × length), 10cm × 25cm (pillar diameter × length), 15cm × 25cm (pillar
Diameter × length).
In some embodiments of the invention, in purification process provided by the invention, Sa Molu peptide sample to be purified is
The Sa Molu peptide of chemical synthesis synthesis.In other embodiment of the invention, in purification process provided by the invention, to
The synthetic method of the Sa Molu peptide sample of purifying is the following steps are included: first synthesizing amino acid resin, gradually coupling synthesis is led later
Then chain peptide resin removes the protecting group of Lys side chain, then be gradually coupled the amino acid and other groups of side chain, finally cutting tree
Rouge obtains the thick peptide of Sa Molu peptide, that is, obtains Sa Molu peptide sample to be purified.
In some embodiments of the invention, it in purification process provided by the invention, is taken in the first separation process to be purified
Sa Molu peptide sample carry out loading before further include with acetonitrile solution rinse chemical bonding silica gel chromatographic column.Of the invention
In some embodiments, in purification process provided by the invention, in the first separation process, when rinsing chemical bonding silica gel chromatographic column,
The percent by volume of acetonitrile is 50%~80% in acetonitrile solution used.
In other embodiment of the invention, in purification process provided by the invention, is taken in the second separation process
Purified carries out further including rinsing chemical bonding silica gel chromatographic column with acetonitrile solution before loading.It is of the invention in addition
In some embodiments, in purification process provided by the invention, in the second separation process, when rinsing chemical bonding silica gel chromatographic column,
The percent by volume of acetonitrile is 50%~80% in acetonitrile solution used.
Preferably, in purification process provided by the invention, further include the steps that into salt, which specifically includes:
Take gained Sa Molu peptide solution, to be chemically bonded silica gel chromatographic column as stationary phase, after loading, through at salt, elute,
Collect elution fraction, get Sa Molu peptide salt；
Wherein, the mixed solution for eluting that mobile phase used is water and acetonitrile for gradient elution, acetonitrile in the solution are eluted
Volume fraction be gradually increased.
In some embodiments of the invention, in purification process provided by the invention, in salt-forming steps, it is chemically bonded silica gel
Chromatographic column is C8 silica gel chromatographic column or butyl bonded silica gel chromatographic column.In other embodiment of the invention, the present invention is mentioned
In the purification process of confession, in salt-forming steps, chemical bonding silica gel chromatographic column is butyl bonded silica gel chromatographic column.
In some embodiments of the invention, in purification process provided by the invention, chemical bond used in salt-forming steps
Close silica gel chromatographic column specification include:
5cm × 25cm (pillar diameter × length), 10cm × 25cm (pillar diameter × length), 15cm × 25cm (pillar
Diameter × length).
In the present invention, the chemical bonding silica gel chromatographic column used in the process of Sa Molu peptide salt-forming steps, separates with first
Used in the process of chemical bonding silica gel chromatographic column, chemical bonding silica gel chromatographic column can phase used in the second separation process
Together, it can also be different.
In other embodiment of the invention, in purification process provided by the invention, in salt-forming steps, at used in salt
Solution be acetonitrile and ammonium acetate mixed solution.In other embodiment of the invention, purifying side provided by the invention
In method, in salt-forming steps, in the mixed solution at acetonitrile used in salt, ammonium acetate and water, the percentage by volume of acetonitrile is 5%；
The percentage by volume of ammonium acetate is 0.1%~0.4%.In other embodiment of the invention, purifying provided by the invention
In method, in salt-forming steps, the pH value at the mixed solution of acetonitrile used in salt, ammonium acetate and water is pH6.5~pH7.0.
In other embodiment of the invention, in purification process provided by the invention, in salt-forming steps, before loading
Further include the steps that rinsing chemical bonding silica gel chromatographic column with acetonitrile solution.In other embodiment of the invention, this
It invents in the purification process provided, acetonitrile solution used in chemical bonding silica gel chromatographic column is rinsed in salt-forming steps, before loading
The percentage by volume of middle acetonitrile is 50%~80%.
In other embodiment of the invention, in purification process provided by the invention, in salt-forming steps, used in elution
Mobile phase in the Volume fraction of water and acetonitrile be 60%:40% → 40%:60%.
In other embodiment of the invention, in purification process provided by the invention, in salt-forming steps, elution is collected
The Volume fraction of water and acetonitrile is 65%:35% → 45%:55% in mobile phase corresponding to component.
The present invention provides a kind of purification process of Sa Molu peptide.The purification process includes: to take Sa Molu peptide to be purified
Sample, separates through chemical bonding silica gel chromatographic column first, carries out gradient elution with A1 solution and the mixed solution of B solution, collects
Elution fraction obtains first time purified；First time purified is taken, is separated through chemical bonding silica gel chromatographic column second, with A2 solution
Gradient elution is carried out with the mixed solution of B solution, collects elution fraction, get Sa Molu peptide solution；A1 solution is carbonate solution；
A2 solution is phosphate solution；B solution is the mixed solution of acetonitrile and isopropanol, the ratio between volume of acetonitrile and isopropanol for (8:
2)~(9:1).Experimental result confirms that the Sa Molu peptide purity is high that purification process provided by the invention prepares significantly improves
The yield of Sa Molu peptide, and it is easy to operate, it is advantageously implemented the prepare with scale of Sa Molu peptide.
Detailed description of the invention
Fig. 1 shows the qualification result of the thick peptide of Sa Molu peptide in embodiment 1；
Fig. 2 shows the testing result of the HPLC of the thick peptide of Sa Molu peptide in embodiment 1, wherein peak T is Sa Molu peptide；
Fig. 3 shows the Mass Spectrometer Method result of gained white powdery solids in embodiment 1；
Fig. 4 shows the testing result of the HPLC of gained Sa Molu peptide salt in embodiment 1；
Fig. 5 shows the testing result of the HPLC of gained Sa Molu peptide salt in embodiment 2；
Fig. 6 shows the testing result of the HPLC of gained Sa Molu peptide salt in embodiment 3；
Fig. 7 shows the testing result of the HPLC of gained Sa Molu peptide salt in embodiment 4；
Fig. 8 shows the testing result of the HPLC of gained Sa Molu peptide salt in embodiment 5；
Fig. 9 shows the testing result of the HPLC of gained Sa Molu peptide salt in embodiment 6；
Figure 10 shows the testing result of the HPLC of gained Sa Molu peptide salt in embodiment 7；
Figure 11 shows the testing result of the HPLC of gained Sa Molu peptide salt in embodiment 8；
Figure 12 shows the testing result of the HPLC of gained Sa Molu peptide salt in comparative example.
Specific embodiment
The invention discloses a kind of purification process of Sa Molu peptide.Those skilled in the art can refer to present disclosure, real
Apply this method, it is accordingly required in particular to, it is noted that all similar substitutions and modifications are apparent for a person skilled in the art
, they are considered as being included in the present invention.Purification process of the invention is described by preferred embodiment,
Related personnel can obviously not depart from the content of present invention, be modified in spirit and scope to methods herein and application or suitably
Changes and combinations carry out implementation and application the technology of the present invention.
Used reagent and raw material are available on the market in a kind of purification process of Sa Molu peptide provided by the invention.
In order to make those skilled in the art better understood when technical solution of the present invention, below with reference to implementation
Example, the present invention is further explained:
The purifying of the thick peptide of 1: Sa Molu peptide of embodiment
Experimental material source: chromatographic column brand Kromasil；Ammonium hydrogen carbonate is purchased from Sinopharm Chemical Reagent Co., Ltd.；
Tetramethylammonium hydroxide is purchased from Aladdin；Sodium dihydrogen phosphate is purchased from Sinopharm Chemical Reagent Co., Ltd.；Ammonium hydroxide is purchased from Guangdong
Brilliance Science and Technology Co., Ltd.；Glacial acetic acid is purchased from Guangdong Guanghua Science and Technology Co., Ltd..
Butyl bonded silica gel chromatographic column: Kromasil--C4-10μm；
The source of the thick peptide of Sa Molu peptide (Sa Molu peptide sample i.e. to be purified): company voluntarily synthesizes.
Synthetic method specifically:
It uses Fmoc-Gly- Wang Shuzhi for initial resin, is added in solid phase reaction column, is washed 2 times with DMF, be swollen with DMF
After Fmoc-Gly- Wang Shuzhi 30 minutes, with DBLK removing Fmoc protection, then washed 4 times with DMF, DCM is washed 2 times, uses ninhydrin
Method detects color of resin, and resin has color, indicates that Fmoc has been removed.Weigh suitable Fmoc-Arg (Pbf)-OH, HOBt
(1.8mmol), PyBOP are dissolved in DCM the and DMF mixed solution that volume ratio is 1 ︰ 1, and DIPEA is added under ice-water bath and activates 3min
It is added in solid phase reaction column, reacts at room temperature 2 hours afterwards.Reaction end is judged with ninhydrin method detection, if resin is colorless and transparent,
Then indicate fully reacting, this judgment criteria judges reaction end suitable for subsequent content with ninhydrin method detection.
It repeats above-mentioned removing Fmoc protection and the step of corresponding amino acid is coupled is added, according to Sa Molu peptide backbone
Peptide sequence is sequentially completed Fmoc-Gly-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp
(Boc)-OH、Fmoc-Ala-OH、Fmoc-Ile-OH、Fmoc-Phe-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Lys
(Alloc)-OH、Fmoc-Ala-OH、Fmoc-Ala-OH、Fmoc-Gln(Trt)-OH、Fmoc-Gly-OH、Fmoc-Glu
(OtBu)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Ser(tBu)-OH、
Fmoc-Val-OH、Fmoc-Asp(OtBu)-OH、Fmoc-Ser(tBu)-OH、Fmoc-Thr(tBu)-OH、Fmoc-Phe-OH、
Fmoc-Thr (tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Ala-OH and Boc-His (Trt)-OH
Coupling.
Methylene chloride is solvent, adds appropriate phenyl silane, reacts 3 minutes, adds Pd (PPh3)4, room temperature reaction 45
Minute, reaction solution is taken out, detects color of resin with ninhydrin method, resin has color, indicates that Alloc has been removed.
Appropriate Fmoc-AEEA-OH, PyBOP, HOBt are weighed, is dissolved with DMF, DIPEA is added under ice-water bath and activates 3 minutes,
Reaction column is added to react 2 hours, reaction end is judged with ninhydrin method detection.Reaction terminates, and DBLK removes Fmoc, DMF washing 6
It is secondary.Same method is coupled Fmoc-AEEA-OH, Fmoc-Glu-OtBu, octadecane diacid again, is received after reaction with methanol
Contracting, is dried in vacuum overnight, obtains peptide resin.
Obtained peptide resin, is added in reaction kettle, by the volume of 5 ︰ of TFA ︰ benzene first Liu Mi ︰ Ben Jia Mi ︰ EDT=90 ︰, 2 ︰ 3
Than preparing lytic reagent, lytic reagent is poured into peptide resin, is reacted at room temperature 2.5 hours.Reaction terminates, and filters resin, collects
Filtrate.Resin is washed with TFA, filtrate is added in anhydrous ether and precipitates by merging filtrate, centrifugation, anhydrous ether washing, and
Vacuum drying, obtains thick peptide.
The qualification result of the thick peptide of Sa Molu peptide is shown in Fig. 1；Its HPLC testing result is shown in Fig. 2, and peak T is Sa Molu peptide in figure, with
External standard method calculates, and wherein the content of Sa Molu peptide is 52%.
By the mixed solution of the thick peptide 2.0g acetonitrile of Sa Molu peptide, ammonium hydroxide and water (volume ratio of acetonitrile, ammonium hydroxide and water=
It 15:5:80) dissolves, filter, it is spare to collect filtrate.
1, step 1: the first separation
Purification condition: chromatographic column: using butyl bonded silica gel chromatographic column as the chromatographic column of stationary phase, pillar diameter and length
Are as follows: 5cm × 25cm.Mobile phase: A1 phase: 50mmol/L ammonium bicarbonate soln solution, with tetramethylammonium hydroxide tune pH to 7.5；B
Phase: trifluoroacetic acid aqueous solution and isopropyl alcohol mixture (the ratio between volume of acetonitrile and isopropanol is 9:1).Flow velocity: 60-80mL/min.
Detection wavelength: 230nm.Gradient: B%:38% → 50%, 50-70min.
Purification process: chromatographic column is rinsed well rear loading with 50% acetonitrile solution, applied sample amount is by 2g Sa Molu peptide
Filtrate made of thick peptide.It after loading, is washed, linear gradient elution 60min.Purpose peak is collected, purpose peak is corresponding
Elution fraction are as follows: B%:44% → 48%.By the peptide solution of collection, vacuum rotary steam is concentrated under conditions of water temperature is no more than 32 DEG C
To about 15mg/mL to get first time purified, obtain first time purified 0.82g altogether (with the content meter of wherein peptide).
2, step 2: the second separation
Purification condition: using butyl bonded silica gel chromatographic column as the chromatographic column of stationary phase, pillar diameter and length are as follows: 5cm ×
25cm.Mobile phase: A2 phase: 50mmol/L sodium dihydrogen phosphate ammonium hydroxide tune pH to 2.0；B phase: trifluoroacetic acid aqueous solution and isopropanol
Mixed solution (the ratio between volume of acetonitrile and isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient:
B%:40% → 50%, 50-70min.
Purification process: chromatographic column is rinsed well rear loading with 50% acetonitrile, applied sample amount is the first step resulting first
Secondary purified 0.82g (with the content meter of wherein peptide).It after loading, is washed, linear gradient elution 60min.Collect mesh
Peak, the corresponding elution fraction in purpose peak are as follows: B%:46% → 49%.By the peptide solution of collection in item of the water temperature no more than 32 DEG C
Vacuum rotary steam is concentrated into about 15mg/mL to get Sa Molu peptide solution under part, obtains the Sa Molu peptide of the peptide of Sa Molu containing 0.55g altogether
Solution.
3, turn salt: rinsing butyl bonded silica gel chromatographic column well rear loading with 50% acetonitrile solution, applied sample amount be containing
The Sa Molu peptide solution of 0.55g Sa Molu peptide rinses 30min with 0.1% ammonium acetate solution (pH 6.5) containing 5% acetonitrile.
Finally carry out gradient elution with water and acetonitrile, wherein acetonitrile gradient: B%:40% → 60%, 40min collect purpose peak, purpose
The corresponding elution fraction in peak are as follows: B%:35% → 55%.The purpose peptide solution of collection is no more than at 32 DEG C in water temperature and depressurizes rotation
Inspissation goes to suitable size cillin bottle after being reduced to about 40mg/mL, is freeze-dried later, obtains white powdery solids 0.41g.
Mass Spectrometer Method is carried out to gained white powdery solids, qualification result is shown in Fig. 3；Carry out HPLC detection, testing result
See Fig. 4.It is found that the purity of Sa Molu peptide is 99.38% from HPLC chromatogram, single impurity is respectively less than 0.15%.Purifying is received
Rate be 41% (calculation method are as follows: the quality of Sa Molu peptide in the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide ×
100%)；Total recovery is 20.5%, (calculation method are as follows: the quality of gained Sa Molu peptide salt/Sa Molu peptide thick peptide quality ×
100%).
The thick peptide purification of 2: Sa Molu peptide of embodiment
Experimental material source: chromatographic column brand Kromasil；Saleratus is purchased from Sinopharm Chemical Reagent Co., Ltd.；
Tetramethylammonium hydroxide is purchased from Aladdin；Potassium dihydrogen phosphate is purchased from Sinopharm Chemical Reagent Co., Ltd.；Ammonium hydroxide is purchased from Guangdong
Brilliance Science and Technology Co., Ltd.；Glacial acetic acid is purchased from Guangdong Guanghua Science and Technology Co., Ltd..
Butyl bonded silica gel chromatographic column: Kromasil--C4-10μm；
The source of the thick peptide of Sa Molu peptide (Sa Molu peptide sample i.e. to be purified): same as Example 1, wherein Sa Molu peptide
Content be 52%.
By the mixed solution of the thick peptide 2.0g acetonitrile of Sa Molu peptide, water and ammonium hydroxide (volume ratio of acetonitrile, ammonium hydroxide and water=
It 15:5:80) dissolves, filter, it is spare to collect filtrate.
1, step 1: the first separation
Purification condition: chromatographic column: using butyl bonded silica gel as the chromatographic column of stationary phase, pillar diameter and length are as follows: 5cm ×
25cm.Mobile phase: A1 phase: 50mmol/L potassium bicarbonate solution solution, with tetramethylammonium hydroxide tune pH to 7.5；B phase: chromatography
Pure acetonitrile and isopropyl alcohol mixture (the ratio between volume of acetonitrile and isopropanol is 8:2).Flow velocity: 60-80mL/min.Detect wave
It is long: 230nm.Gradient: B%:38% → 50%, 50-70min.
Purification process: chromatographic column is rinsed well rear loading with 80% acetonitrile solution, applied sample amount is by 2g Sa Molu peptide
Filtrate made of thick peptide.It after loading, is washed, linear gradient elution 60min.Purpose peak is collected, purpose peak is corresponding
Elution fraction are as follows: B%:44% → 48%.By the peptide solution of collection, vacuum rotary steam is concentrated under conditions of water temperature is no more than 32 DEG C
To about 15mg/mL to get first time purified, obtain first time purified 0.88g altogether (with the content meter of wherein peptide).
2, step 2: the second separation
Purification condition: chromatographic column: using butyl bonded silica gel as the chromatographic column of stationary phase, pillar diameter and length are as follows: 5cm ×
25cm.Mobile phase: A2 phase: 20mmol/L potassium dihydrogen phosphate ammonium hydroxide tune pH to 3.0；B phase: trifluoroacetic acid aqueous solution and isopropanol
Mixed solution (the ratio between volume of acetonitrile and isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient:
B%:40% → 50%, 50-70min.
Purification process: chromatographic column is rinsed well rear loading with 80% acetonitrile, applied sample amount is the first step resulting first
Secondary purified 0.88g (with the content meter of wherein peptide).It after loading, is washed, linear gradient elution 60min.Collect mesh
Peak, the corresponding elution fraction in purpose peak are as follows: B%:46% → 49%.By the peptide solution of collection in item of the water temperature no more than 32 DEG C
Vacuum rotary steam is concentrated into about 15mg/mL to get Sa Molu peptide solution under part, and the Sa Molu peptide for obtaining the peptide of Sa Molu containing 0.5g altogether is molten
Liquid.
3, turn salt: rinsing butyl bonded silica gel chromatographic column well rear loading with 80% acetonitrile solution, applied sample amount be containing
The Sa Molu peptide solution of 0.5g Sa Molu peptide.15min is rinsed with 0.4% ammonium acetate solution (pH 7.0) containing 5% acetonitrile.
Finally use gradient elution, carry out gradient elution with water and acetonitrile, wherein acetonitrile gradient: B%:40% → 60%, 40min are collected
Purpose peak, the corresponding elution fraction in purpose peak are as follows: B%:35% → 55%.The purpose peptide solution of collection is no more than 32 in water temperature
Vacuum rotary steam goes to suitable size cillin bottle after being concentrated into about 45mg/mL at DEG C, is freeze-dried later, obtains white powder
Solid 0.4g.
HPLC detection is carried out to gained white powdery solids, testing result is shown in Fig. 5, and purity 99.25% is single miscellaneous
Matter is respectively less than 0.15%.Purifying yield is 40% (calculation method are as follows: in the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide
Quality × 100% of Sa Molu peptide)；Total recovery is 20.2%, (calculation method are as follows: quality/Sa Molu of gained Sa Molu peptide salt
Quality × 100% of the thick peptide of peptide).
The thick peptide purification of 3: Sa Molu peptide of embodiment
Experimental material source: chromatographic column brand Kromasil；Ammonium carbonate is purchased from Sinopharm Chemical Reagent Co., Ltd.；Four
Ammonium hydroxide is purchased from Aladdin；Potassium dihydrogen phosphate is purchased from Sinopharm Chemical Reagent Co., Ltd.；Ammonium hydroxide is purchased from Guangdong light
Magnificent Science and Technology Co., Ltd.；Glacial acetic acid is purchased from Guangdong Guanghua Science and Technology Co., Ltd..
Butyl bonded silica gel chromatographic column: Kromasil--C4-10μm；
The source of the thick peptide of Sa Molu peptide (Sa Molu peptide sample i.e. to be purified): same as Example 1, wherein Sa Molu peptide
Content be 52%.
By the thick peptide 2.0g dissolution of Sa Molu peptide, filtering, it is spare to collect filtrate.
1, step 1: the first separation
Purification condition: chromatographic column: using butyl bonded silica gel as the chromatographic column of stationary phase, pillar diameter and length are as follows: 5cm ×
25cm.Mobile phase: A1 phase: 20mmol/L sal volatile, with tetramethylammonium hydroxide tune pH to 8.5；B phase: trifluoroacetic acid aqueous solution
With isopropyl alcohol mixture (the ratio between volume of acetonitrile and isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength:
230nm.Gradient: B%:38% → 50%, 50-70min.
Purification process: chromatographic column is rinsed well rear loading with 75% acetonitrile solution, applied sample amount is by 2g Sa Molu peptide
Filtrate made of thick peptide.It after loading, is washed, linear gradient elution 60min.Purpose peak is collected, purpose peak is corresponding
Elution fraction are as follows: B%:44% → 48%.By the peptide solution of collection, vacuum rotary steam is concentrated under conditions of water temperature is no more than 32 DEG C
To about 15mg/mL to get first time purified, obtain first time purified 0.75g altogether (with the content meter of wherein peptide).
2, step 2: the second separation
Purification condition: chromatographic column: using butyl bonded silica gel as the chromatographic column of stationary phase, pillar diameter and length are as follows: 5cm ×
25cm.Mobile phase: A2 phase: 50mmol/L potassium dihydrogen phosphate ammonium hydroxide tune pH to 2.0；B phase: trifluoroacetic acid aqueous solution and isopropanol
Mixed solution (the ratio between volume of acetonitrile and isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient:
B%:40% → 50%, 50-70min.
Purification process: chromatographic column is rinsed well rear loading with 75% acetonitrile solution, applied sample amount is that the first step is resulting
First time purified 0.75g (with the content meter of wherein peptide).It after loading, is washed, linear gradient elution 60min.It receives
Ji Mufeng, the corresponding elution fraction in purpose peak are as follows: B%:46% → 49%.The peptide solution of collection is no more than 32 DEG C in water temperature
Under conditions of vacuum rotary steam be concentrated into about 15mg/mL to get Sa Molu peptide solution, obtain the Sa Mo of the peptide of Sa Molu containing 0.62g altogether
Shandong peptide solution.It carries out turning salt.
3, turn salt: rinsing butyl bonded silica gel chromatographic column well rear loading with 75% acetonitrile solution, applied sample amount be containing
The Sa Molu peptide solution of 0.62g Sa Molu peptide.20min is rinsed with 0.3% ammonium acetate solution (pH 6.8) containing 5% acetonitrile.
Finally use gradient elution, carry out gradient elution with water and acetonitrile, wherein acetonitrile gradient: B%:40% → 60%, 40min are collected
Purpose peak, the corresponding elution fraction in purpose peak are as follows: B%:35% → 55%.The purpose peptide solution of collection is no more than 32 in water temperature
Vacuum rotary steam goes to suitable size cillin bottle after being concentrated into about 45mg/mL at DEG C, is freeze-dried later, obtains white powder
Solid 0.5g.
HPLC detection is carried out to gained white powdery solids, testing result is shown in Fig. 6, and purity 99.38% is single miscellaneous
Matter is respectively less than 0.15%.Purifying yield is 50% (calculation method are as follows: in the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide
Quality × 100% of Sa Molu peptide)；Total recovery is 25%, (calculation method are as follows: quality/Sa Molu peptide of gained Sa Molu peptide salt
Quality × 100% of thick peptide).
The thick peptide purification of 4: Sa Molu peptide of embodiment
Experimental material source: chromatographic column brand Kromasil；Ammonium hydrogen carbonate is purchased from Sinopharm Chemical Reagent Co., Ltd.；
Tetramethylammonium hydroxide is purchased from Aladdin；Sodium dihydrogen phosphate is purchased from Sinopharm Chemical Reagent Co., Ltd.；Ammonium hydroxide is purchased from Guangdong
Brilliance Science and Technology Co., Ltd.；Glacial acetic acid is purchased from Guangdong Guanghua Science and Technology Co., Ltd..
Butyl bonded silica gel chromatographic column: Kromasil--C4-10μm；
The source of the thick peptide of Sa Molu peptide (Sa Molu peptide sample i.e. to be purified): same as Example 1, wherein Sa Molu peptide
Content be 52%.
By the mixed solution of the thick peptide 2.0g acetonitrile of Sa Molu peptide, water and ammonium hydroxide (volume ratio of acetonitrile, ammonium hydroxide and water=
It 15:5:80) dissolves, filter, it is spare to collect filtrate.
1, step 1: the first separation
Purification condition: chromatographic column: using butyl bonded silica gel as the chromatographic column of stationary phase, pillar diameter and length are as follows: 5cm ×
25cm.Mobile phase: A1 phase: 50mmol/L ammonium bicarbonate soln, with tetramethylammonium hydroxide tune pH to 8.5；B phase: chromatographically pure second
Nitrile and isopropyl alcohol mixture (the ratio between volume of acetonitrile and isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength:
230nm.Gradient: B%:38% → 50%, 50-70min.
Purification process: chromatographic column is rinsed well rear loading with 75% acetonitrile solution, applied sample amount is by 2g Sa Molu peptide
Filtrate made of thick peptide.It after loading, is washed, linear gradient elution 60min.Purpose peak is collected, purpose peak is corresponding
Elution fraction are as follows: B%:44% → 48%.By the peptide solution of collection, vacuum rotary steam is concentrated under conditions of water temperature is no more than 32 DEG C
To about 15mg/mL to get first time purified, obtain first time purified 0.78g altogether (with the content meter of wherein peptide).
Step 2: the second separation
Purification condition: chromatographic column: using butyl bonded silica gel as the chromatographic column of stationary phase, pillar diameter and length are as follows: 5cm ×
25cm.Mobile phase: A2 phase: 50mmol/L sodium dihydrogen phosphate ammonium hydroxide tune pH to 3.0；B phase: trifluoroacetic acid aqueous solution and isopropanol
Mixed solution (the ratio between volume of acetonitrile and isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient:
B%:40% → 50%, 50-70min.
Purification process: chromatographic column is rinsed well rear loading with 75% acetonitrile solution, applied sample amount is that the first step is resulting
First time purified 0.78g (with the content meter of wherein peptide).It after loading, is washed, linear gradient elution 60min.It receives
Ji Mufeng, the corresponding elution fraction in purpose peak are as follows: B%:46% → 49%.The peptide solution of collection is no more than 32 DEG C in water temperature
Under conditions of vacuum rotary steam be concentrated into about 15mg/mL to get Sa Molu peptide solution, obtain the Sa Molu of the peptide of Sa Molu containing 0.6g altogether
Peptide solution carries out turning salt.
3, turn salt: rinsing butyl bonded silica gel chromatographic column well rear loading with 75% acetonitrile solution, applied sample amount be containing
The Sa Molu peptide solution of 0.6g Sa Molu peptide rinses 20min with 0.2% ammonium acetate solution (pH 6.5) containing 5% acetonitrile.
Finally use gradient elution, carry out gradient elution with water and acetonitrile, wherein acetonitrile gradient: B%:40% → 60%, 40min are collected
Purpose peak, the corresponding elution fraction in purpose peak are as follows: B%:35% → 55%.The purpose peptide solution of collection is no more than 32 in water temperature
Vacuum rotary steam goes to suitable size cillin bottle after being concentrated into about 15mg/mL at DEG C, is freeze-dried later, obtains white powder
Solid 0.45g.
HPLC detection is carried out to gained white powdery solids, testing result is shown in Fig. 7, and purity 99.30% is single miscellaneous
Matter is respectively less than 0.15%.Purifying yield is 45% (calculation method are as follows: in the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide
Quality × 100% of Sa Molu peptide)；Total recovery is 22.5%, (calculation method are as follows: quality/Sa Molu of gained Sa Molu peptide salt
Quality × 100% of the thick peptide of peptide).
The thick peptide purification of 5: Sa Molu peptide of embodiment
Experimental material source: chromatographic column brand Kromasil；Ammonium hydrogen carbonate is purchased from Sinopharm Chemical Reagent Co., Ltd.；
Tetramethylammonium hydroxide is purchased from Aladdin；Potassium dihydrogen phosphate is purchased from Sinopharm Chemical Reagent Co., Ltd.；Ammonium hydroxide is purchased from Guangdong
Brilliance Science and Technology Co., Ltd.；Glacial acetic acid is purchased from Guangdong Guanghua Science and Technology Co., Ltd..
Butyl bonded silica gel chromatographic column: Kromasil--C4-10μm；
The source of the thick peptide of Sa Molu peptide (Sa Molu peptide sample i.e. to be purified): same as Example 1, wherein Sa Molu peptide
Content be 52%.
By the mixed solution of the thick peptide 2.0g acetonitrile of Sa Molu peptide, water and ammonium hydroxide (volume ratio of acetonitrile, ammonium hydroxide and water=
It 15:5:80) dissolves, filter, it is spare to collect filtrate.
1, step 1: the first separation
Purification condition: chromatographic column: using butyl bonded silica gel as the chromatographic column of stationary phase, pillar diameter and length are as follows: 5cm ×
25cm.Mobile phase: 1 phase of A: 150mmol/L ammonium bicarbonate soln solution, with tetramethylammonium hydroxide tune pH to 8.0；B phase: color
Compose pure acetonitrile and isopropyl alcohol mixture (the ratio between volume of acetonitrile and isopropanol is 9:1).Flow velocity: 60-80mL/min.Detect wave
It is long: 230nm.Gradient: B%:38% → 50%, 50-70min.
Chromatographic column is rinsed well rear loading with 75% acetonitrile solution, applied sample amount is made of the thick peptide of 2g Sa Molu peptide
Filtrate.It after loading, is washed, linear gradient elution 60min.Collect purpose peak, the corresponding elution fraction in purpose peak are as follows:
B%:44% → 48%.By the peptide solution of collection, vacuum rotary steam is concentrated into about 15mg/mL under conditions of water temperature is no more than 32 DEG C,
Up to first time purified, obtain first time purified 0.8g altogether (with the content meter of wherein peptide).
2, step 2: the second separation
Purification condition: chromatographic column: using butyl bonded silica gel as the chromatographic column of stationary phase, pillar diameter and length are as follows: 5cm ×
25cm.Mobile phase: A2 phase: 100mmol/L dipotassium hydrogen phosphate solution ammonium hydroxide tune pH to 2.5；B phase: trifluoroacetic acid aqueous solution and isopropyl
The mixed solution of alcohol (the ratio between volume of ethyl alcohol and isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Ladder
Degree: B%:40% → 50%, 50-70min.
Purification process: chromatographic column is rinsed well rear loading with 50% acetonitrile solution, applied sample amount is that the first step is resulting
First time purified 0.8g (with the content meter of wherein peptide).It after loading, is washed, linear gradient elution 60min.It collects
Purpose peak, the corresponding elution fraction in purpose peak are as follows: B%:46% → 49%.The peptide solution of collection is no more than 32 DEG C in water temperature
Under the conditions of vacuum rotary steam be concentrated into about 15mg/mL to get Sa Molu peptide solution, obtain the Sa Molu of the peptide of Sa Molu containing 0.57g altogether
Peptide solution carries out turning salt.
3, turn salt: rinsing butyl bonded silica gel chromatographic column well rear loading with 50% acetonitrile solution, applied sample amount be containing
The Sa Molu peptide solution of 0.57g Sa Molu peptide rinses 30min with 0.4% ammonium acetate solution (pH 6.5) containing 5% acetonitrile.
Last gradient elution carries out gradient elution with water and acetonitrile, wherein acetonitrile gradient: B%:40% → 60%, 40min, collects mesh
Peak, the corresponding elution fraction in purpose peak are as follows: B%:35% → 55%.The purpose peptide solution of collection is no more than 32 DEG C in water temperature
Lower vacuum rotary steam goes to suitable size cillin bottle after being concentrated into about 15mg/mL, is freeze-dried later, and it is solid to obtain white powder
Body 0.5g.
HPLC detection is carried out to gained white powdery solids, testing result is shown in Fig. 8, and purity 99.20% is single miscellaneous
Matter is respectively less than 0.15%.Purifying yield is 50% (calculation method are as follows: in the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide
Quality × 100% of Sa Molu peptide)；Total recovery is 25%, (calculation method are as follows: quality/Sa Molu peptide of gained Sa Molu peptide salt
Quality × 100% of thick peptide).
The thick peptide purification of 6: Sa Molu peptide of embodiment
Experimental material source: chromatographic column brand Kromasil；Ammonium hydrogen carbonate is purchased from Sinopharm Chemical Reagent Co., Ltd.；
Tetramethylammonium hydroxide is purchased from Aladdin；Sodium dihydrogen phosphate is purchased from Sinopharm Chemical Reagent Co., Ltd.；Ammonium hydroxide is purchased from Guangdong
Brilliance Science and Technology Co., Ltd.；Glacial acetic acid is purchased from Guangdong Guanghua Science and Technology Co., Ltd..
Butyl bonded silica gel chromatographic column: Kromasil--C4-10μm；
The source of the thick peptide of Sa Molu peptide (Sa Molu peptide sample i.e. to be purified): same as Example 1, wherein Sa Molu peptide
Content be 52%.
By the mixed solution of the thick peptide 15g acetonitrile of Sa Molu peptide, water and ammonium hydroxide (volume ratio=15 of acetonitrile, ammonium hydroxide and water:
It 5:80) dissolves, filter, it is spare to collect filtrate.
1, step 1: the first separation
Purification condition: chromatographic column: using butyl bonded silica gel as the chromatographic column of stationary phase, pillar diameter and length are as follows: 10cm
×25cm.Mobile phase: A1 phase: 100mmol/L ammonium bicarbonate soln solution, with tetramethylammonium hydroxide tune pH to 8.0；B phase: color
Compose the mixed solution of pure acetonitrile and isopropanol (the ratio between volume of acetonitrile and isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection
Wavelength: 230nm.Gradient: B%:38% → 50%, 50-70min.
Purification process: chromatographic column is rinsed well rear loading with 75% acetonitrile solution, applied sample amount is by 15g Sa Molu peptide
Filtrate made of thick peptide.It after loading, is washed, linear gradient elution 60min.Purpose peak is collected, purpose peak is corresponding
Elution fraction are as follows: B%:44% → 48%.By the peptide solution of collection, vacuum rotary steam is concentrated under conditions of water temperature is no more than 32 DEG C
To about 15mg/mL to get first time purified, obtain first time purified 6g altogether (with the content meter of wherein peptide).
2, step 2: the second separation
Purification condition: chromatographic column: using butyl bonded silica gel as the chromatographic column of stationary phase, pillar diameter and length are as follows: 10cm
×25cm.Mobile phase: A2 phase: 100mmol/L sodium dihydrogen phosphate, with ammonium hydroxide tune pH to 2.5；B phase: trifluoroacetic acid aqueous solution with it is different
The mixed solution of propyl alcohol (the ratio between volume of acetonitrile and isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.
Gradient: B%:40% → 50%, 50-70min.
Purification process: chromatographic column is rinsed well rear loading with 80% acetonitrile, applied sample amount is first time obtained by the first step
Purified 6g (with the content meter of wherein peptide).It after loading, is washed, linear gradient elution 60min.Purpose peak is collected,
The corresponding elution fraction in purpose peak are as follows: B%:46% → 49%.By the peptide solution of collection under conditions of water temperature is no more than 32 DEG C
Vacuum rotary steam is concentrated into about 15mg/mL to get Sa Molu peptide solution, obtains the Sa Molu peptide solution of the peptide of Sa Molu containing 4.8g altogether,
It carries out turning salt.
3, turn salt: rinsing butyl bonded silica gel chromatographic column well rear loading with 80% acetonitrile solution, applied sample amount be containing
The Sa Molu peptide of 4.8g Sa Molu peptide is molten, rinses 15min with 0.4% ammonium acetate solution (pH 7.0) containing 5% acetonitrile, most
Use gradient elution afterwards, carry out gradient elution with water and acetonitrile, wherein acetonitrile gradient: B%:40% → 60%, 40min collect mesh
Peak, the corresponding elution fraction in purpose peak are as follows: B%:35% → 55%.The purpose peptide solution of collection is no more than 32 DEG C in water temperature
Lower vacuum rotary steam goes to suitable size cillin bottle after being concentrated into about 45mg/mL.It is freeze-dried later, it is solid to obtain white powder
Body 4.2g.
HPLC detection is carried out to gained white powdery solids, testing result is shown in Fig. 9, and purity 99.38% is single miscellaneous
Matter is respectively less than 0.15%.Purifying yield is 53.8% (calculation method are as follows: the quality of the gained Sa Molu peptide salt/thick peptide of Sa Molu peptide
Quality × 100% of middle Sa Molu peptide)；Total recovery is 28%, (calculation method are as follows: quality/Sa Molu of gained Sa Molu peptide salt
Quality × 100% of the thick peptide of peptide).
The thick peptide purification of 7: Sa Molu peptide of embodiment
Experimental material source: chromatographic column brand Kromasil；Ammonium hydrogen carbonate is purchased from Sinopharm Chemical Reagent Co., Ltd.；
Tetramethylammonium hydroxide is purchased from Aladdin；Potassium dihydrogen phosphate is purchased from Sinopharm Chemical Reagent Co., Ltd.；Ammonium hydroxide is purchased from Guangdong
Brilliance Science and Technology Co., Ltd.；Glacial acetic acid is purchased from Guangdong Guanghua Science and Technology Co., Ltd..
Butyl bonded silica gel chromatographic column: Kromasil--C4-10μm；
The source of the thick peptide of Sa Molu peptide (Sa Molu peptide sample i.e. to be purified): same as Example 1, wherein Sa Molu peptide
Content be 52%.
The thick peptide 25g acetonitrile of Sa Molu peptide, water and ammonium hydroxide (volume ratio=15:5:80 of acetonitrile, ammonium hydroxide and water) are dissolved,
It is spare to collect filtrate for filtering.
1, step 1: the first separation
Purification condition: chromatographic column: using butyl bonded silica gel as the chromatographic column of stationary phase, pillar diameter and length are as follows: 15cm
×25cm.Mobile phase: A1 phase: 100mmol/L ammonium bicarbonate soln solution, with tetramethylammonium hydroxide tune pH to 8.0；B phase: color
Compose pure acetonitrile and isopropyl alcohol mixture (the ratio between volume of ethyl alcohol and isopropanol is 9:1).Flow velocity: 60-80mL/min.Detect wave
It is long: 230nm.Gradient: B%:38% → 50%, 50-70min.
Purification process: chromatographic column is rinsed well rear loading with 75% acetonitrile solution, applied sample amount is by 25g Sa Molu peptide
Filtrate made of thick peptide.It after loading, is washed, linear gradient elution 60min.Purpose peak is collected, purpose peak is corresponding
Elution fraction are as follows: B%:44% → 48%.By the peptide solution of collection, vacuum rotary steam is concentrated under conditions of water temperature is no more than 32 DEG C
To about 15mg/mL to get first time purified, obtain first time purified 10g altogether (with the content meter of wherein peptide).
2, step 2: the second separation
Purification condition: chromatographic column: using butyl bonded silica gel as the chromatographic column of stationary phase, pillar diameter and length are as follows: 15cm
×25cm.Mobile phase: A2 phase: 150mmol/L potassium dihydrogen phosphate ammonium hydroxide tune pH to 2.5；B phase: trifluoroacetic acid aqueous solution and different
Propyl alcohol mixed solution (the ratio between volume of acetonitrile and isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Ladder
Degree: B%:40% → 50%, 50-70min.Sample volume is 20-30g.
Purification process: chromatographic column is rinsed well rear loading with 60% acetonitrile, applied sample amount is first time obtained by the first step
Purified 10g (with the content meter of wherein peptide).It after loading, is washed, linear gradient elution 60min.Purpose peak is collected,
The corresponding elution fraction in purpose peak are as follows: B%:46% → 49%.By the peptide solution of collection under conditions of water temperature is no more than 32 DEG C
Vacuum rotary steam is concentrated into about 15mg/mL to get Sa Molu peptide solution, obtains the Sa Molu peptide solution of the peptide of Sa Molu containing 8.1g altogether,
It carries out turning salt.
3, turn salt: rinsing butyl bonded silica gel chromatographic column well rear loading with 60% acetonitrile solution, applied sample amount be containing
The Sa Molu peptide solution of 8.1g Sa Molu peptide rinses 30min with 0.4% ammonium acetate solution (pH 6.8) containing 5% acetonitrile,
Finally use gradient elution, carry out gradient elution with water and acetonitrile, wherein acetonitrile gradient: B%:40% → 60%, 40min are collected
Purpose peak, the corresponding elution fraction in purpose peak are as follows: B%:35% → 55%.The purpose peptide solution of collection is no more than 32 in water temperature
Vacuum rotary steam goes to suitable size cillin bottle after being concentrated into about 40mg/mL under conditions of DEG C.It is freeze-dried, is obtained white later
Pulverulent solids 7.4g.
HPLC detection is carried out to gained white powdery solids, testing result is shown in Figure 10, purity 99.45%, individually
Impurity is respectively less than 0.15%.Purifying yield is 56% (calculation method are as follows: the weight of the gained Sa Molu peptide salt/thick peptide of Sa Molu peptide
Quality × 100% of middle Sa Molu peptide)；Total recovery is 29.6%, (calculation method are as follows: quality/Sa Mo of gained Sa Molu peptide salt
Quality × 100% of the thick peptide of Shandong peptide).
The thick peptide purification of 8: Sa Molu peptide of embodiment
Experimental material source: chromatographic column brand Kromasil；Ammonium hydrogen carbonate is purchased from Sinopharm Chemical Reagent Co., Ltd.；
Tetramethylammonium hydroxide is purchased from Aladdin；Sodium dihydrogen phosphate is purchased from Sinopharm Chemical Reagent Co., Ltd.；Ammonium hydroxide is purchased from Guangdong
Brilliance Science and Technology Co., Ltd.；Glacial acetic acid is purchased from Guangdong Guanghua Science and Technology Co., Ltd..
C8 silica gel chromatographic column: Kromasil--C8-10μm；
The source of the thick peptide of Sa Molu peptide (Sa Molu peptide sample i.e. to be purified): same as Example 1, wherein Sa Molu peptide
Content be 52%.
The thick peptide 15g acetonitrile of Sa Molu peptide, water and ammonium hydroxide (volume ratio=15:5:80 of acetonitrile, ammonium hydroxide and water) are dissolved,
It is spare to collect filtrate for filtering.
1, step 1: the first separation
Purification condition: chromatographic column: using C8 silica gel chromatographic column as the chromatographic column of stationary phase, pillar diameter and length are as follows: 10cm
×25cm.Mobile phase: A1 phase: 100mmol/L ammonium bicarbonate soln solution, with tetramethylammonium hydroxide tune pH to 8.0；B phase: color
Compose the mixed solution of pure acetonitrile and isopropanol (the ratio between volume of acetonitrile and isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection
Wavelength: 230nm.Gradient: B%:38% → 50%, 50-70min.
Purification process: chromatographic column is rinsed well rear loading with 75% acetonitrile solution, applied sample amount is by 15g Sa Molu peptide
Filtrate made of thick peptide.It after loading, is washed, linear gradient elution 60min.Purpose peak is collected, purpose peak is corresponding
Elution fraction are as follows: B%:44% → 48%.By the peptide solution of collection, vacuum rotary steam is concentrated under conditions of water temperature is no more than 32 DEG C
To about 15mg/mL to get first time purified, obtain first time purified 5g altogether (with the content meter of wherein peptide).
2, step 2: the second separation
Purification condition: chromatographic column: using C8 silica gel chromatographic column as the chromatographic column of stationary phase, pillar diameter and length are as follows: 10cm
×25cm.Mobile phase: A2 phase: 20mmol/L sodium dihydrogen phosphate, with ammonium hydroxide tune pH to 2.5；B phase: trifluoroacetic acid aqueous solution with it is different
The mixed solution of propyl alcohol (the ratio between volume of acetonitrile and isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.
Gradient: B%:40% → 50%, 50-70min.
Purification process: chromatographic column is rinsed well rear loading with 80% acetonitrile, applied sample amount is first obtained by the first step
Secondary purified 5g (with the content meter of wherein peptide).It after loading, is washed, linear gradient elution 60min.Finally, carrying out
Elution, linear gradient elution 60min collect purpose peak, the corresponding elution fraction in purpose peak are as follows: B%:46% → 49%.It will receive
The peptide solution collected vacuum rotary steam under conditions of water temperature is no more than 32 DEG C is concentrated into about 15mg/mL to get Sa Molu peptide solution, altogether
The Sa Molu peptide solution of the peptide of Sa Molu containing 3.2g is obtained, it is spare.
3, turn salt: rinsing C8 silica gel chromatographic column column well rear loading with 80% acetonitrile solution, applied sample amount is containing 3.2g
The Sa Molu peptide solution of Sa Molu peptide rinses 15min with 0.4% ammonium acetate solution (pH 7.0) containing 5% acetonitrile, finally
With gradient elution, carry out gradient elution with water and acetonitrile, wherein acetonitrile gradient: B%:40% → 60%, 40min collect purpose
Peak, the corresponding elution fraction in purpose peak are as follows: B%:35% → 55%.The purpose peptide solution of collection is no more than at 32 DEG C in water temperature
Vacuum rotary steam goes to suitable size cillin bottle after being concentrated into about 45mg/mL.It is freeze-dried later, obtains white powdery solids
2.3g。
HPLC detection is carried out to gained white powdery solids, testing result is shown in Figure 11, and purity 99.28%% is single
A impurity is respectively less than 0.15%.Purifying yield is 29% (calculation method are as follows: quality/Sa Molu peptide of gained Sa Molu peptide salt is thick
Quality × 100% of Sa Molu peptide in peptide)；Total recovery is 15.3%, (calculation method are as follows: quality/Sa of gained Sa Molu peptide salt
Rub quality × 100% of the thick peptide of Shandong peptide).
Comparative example
Experimental material source: chromatographic column brand Kromasil；Ammonium hydrogen carbonate is purchased from Sinopharm Chemical Reagent Co., Ltd.；
Tetramethylammonium hydroxide is purchased from Aladdin；Sodium dihydrogen phosphate is purchased from Sinopharm Chemical Reagent Co., Ltd.；Ammonium hydroxide is purchased from Guangdong
Brilliance Science and Technology Co., Ltd.；Glacial acetic acid is purchased from Guangdong Guanghua Science and Technology Co., Ltd..
Octadecyl silane: Kromasil--C18-10μm；
The source of the thick peptide of Sa Molu peptide (Sa Molu peptide sample i.e. to be purified): same as Example 1, wherein Sa Molu peptide
Content be 52%.
The thick peptide 2.0g acetonitrile of Sa Molu peptide, water and ammonium hydroxide (volume ratio=15:5:80 of acetonitrile, ammonium hydroxide and water) is molten
Solution, filtering, it is spare to collect filtrate.
1, step 1: the first separation
Purification condition: chromatographic column: using octadecyl silane as the chromatographic column of stationary phase, pillar diameter and length are as follows:
5cm×25cm.Mobile phase: A1 phase: 150mmol/L ammonium bicarbonate soln solution, with tetramethylammonium hydroxide tune pH to 8.0；B
Phase: trifluoroacetic acid aqueous solution and isopropyl alcohol mixture (the ratio between volume of acetonitrile and isopropanol is 9:1).Flow velocity: 60-80mL/min.
Detection wavelength: 230nm.Gradient: B%:40% → 55%, 50-70min.
Purification process: chromatographic column is rinsed well rear loading with 75% acetonitrile solution, applied sample amount is by 2g Sa Molu peptide
Filtrate made of thick peptide.It after loading, is washed, linear gradient elution 60min.Purpose peak is collected, purpose peak is corresponding
Elution fraction are as follows: B%:44% → 48%.By the peptide solution of collection, vacuum rotary steam is concentrated under conditions of water temperature is no more than 32 DEG C
To about 15mg/mL to get first time purified, obtain first time purified 0.5g altogether (with the content meter of wherein peptide).
2, step 2: the second separation
Purification condition: chromatographic column: using octadecyl silane as the chromatographic column of stationary phase, pillar diameter and length are as follows:
5cm×25cm.Mobile phase: A2 phase: 100mmol/L sodium dihydrogen phosphate ammonium hydroxide tune pH to 2.5；B phase: trifluoroacetic acid aqueous solution and
The mixed solution of isopropanol (the ratio between volume of ethyl alcohol and isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength:
230nm.Gradient: B%:42% → 52%, 50-70min.
Purification process: chromatographic column is rinsed well rear loading with 50% acetonitrile solution, applied sample amount is what the first step obtained
0.5g first time purified (with the content meter of peptide therein).It after loading, is washed, linear gradient elution 60min.It receives
Ji Mufeng, the corresponding elution fraction in purpose peak are as follows: B%:46% → 49%.The peptide solution of collection is no more than 32 DEG C in water temperature
Under conditions of vacuum rotary steam be concentrated into about 15mg/mL to get Sa Molu peptide solution, obtain the Sa Molu of the peptide of Sa Molu containing 0.4g altogether
Peptide solution carries out turning salt.
3, turn salt: rinsing butyl bonded silica gel chromatographic column well rear loading with 50% acetonitrile solution, applied sample amount be containing
The Sa Molu peptide solution of 0.4g Sa Molu peptide rinses 30min with 0.4% ammonium acetate solution (pH 6.5) containing 5% acetonitrile.
Last gradient elution carries out gradient elution with water and acetonitrile, wherein acetonitrile gradient: B%:40% → 60%, 40min, collects mesh
Peak, by the purpose peptide solution of collection in water temperature be no more than after vacuum rotary steam is concentrated into about 15mg/mL at 32 DEG C go to it is suitable big
Small cillin bottle, is freeze-dried later, obtains white powdery solids 0.2g.
HPLC detection is carried out to gained white powdery solids, testing result is shown in Figure 12, and purity 98.2% is single miscellaneous
Matter is respectively less than 0.3%.Purifying yield is 20% (calculation method are as follows: Sa in the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide
Rub quality × 100% of Shandong peptide)；Total recovery is 10%, (calculation method are as follows: quality/Sa Molu peptide of gained Sa Molu peptide salt is thick
Quality × 100% of peptide).
The above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
Limitation of the invention, protection scope of the present invention should be defined by the scope defined by the claims..For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, these change
It also should be regarded as protection scope of the present invention into retouching.
Claims (2)
1. a kind of purification process of Sa Molu peptide, characterized in that it comprises:
Sa Molu peptide sample to be purified is taken, is separated through chemical bonding silica gel chromatographic column first, with the mixing of A1 solution and B solution
Solution carries out gradient elution, collects elution fraction, obtains first time purified；
The first time purified is taken, is separated through chemical bonding silica gel chromatographic column second, with the mixed solution of A2 solution and B solution
Gradient elution is carried out, elution fraction, get Sa Molu peptide solution are collected；
The A1 solution is carbonate solution；
The A2 solution is phosphate solution；
The B solution is the mixed solution of acetonitrile and isopropanol, the ratio between volume of the acetonitrile and isopropanol for (8:2)~(9:
1)；
The concentration of the carbonate solution is 20mmol/L~150mmol/L；
The pH value of the A1 solution is 7.5~8.5；
The concentration of the phosphate solution is 20mmol/L~150mmol/L；
The pH value of the A2 solution is 2.0~3.0；
In first separation, in mobile phase used in the gradient elution, the Volume fraction of the A1 solution and the B solution
For 62%:38% → 50%:50%；
In second separation, in mobile phase used in the gradient elution, the Volume fraction of the A2 solution and the B solution
For 60%:40% → 50%:50%；
In second separation, the body for collecting A2 solution and the B solution described in mobile phase corresponding to elution fraction
Fraction ratio is 54%:46% → 51%:49%；
In second separation, the chemical bonding silica gel chromatographic column is C8 silica gel chromatographic column or butyl bonded silica gel chromatographic column.
2. purification process according to claim 1, which is characterized in that further include the steps that into salt, specifically:
The Sa Molu peptide solution is taken, to be chemically bonded silica gel chromatographic column as stationary phase, after loading, through collecting at salt, elution
Elution fraction, get Sa Molu peptide salt；
The elution is gradient elution, and mobile phase used in the elution is the mixed solution of water and acetonitrile, the body of the acetonitrile
Fraction is gradually increased.
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| WO2018032521A1 (en) * | 2016-08-19 | 2018-02-22 | 深圳市健元医药科技有限公司 | Method for synthesizing liraglutide |
| ES2976496T3 (en) | 2017-08-24 | 2024-08-02 | Novo Nordisk As | GLP-1 compositions and uses thereof |
| CN111269309B (en) * | 2018-12-04 | 2022-03-08 | 翰宇药业（武汉）有限公司 | Purification method of GLP-1 analog polypeptide |
| CN109354622A (en) * | 2018-12-05 | 2019-02-19 | 苏州汇通色谱分离纯化有限公司 | A kind of Suo Malu peptide purification filler special and its purification process |
| WO2020190757A1 (en) | 2019-03-15 | 2020-09-24 | Novetide Ltd. | Improved processes for the preparation of semaglutide |
| CN112279907B (en) * | 2019-07-27 | 2023-10-03 | 深圳市健元医药科技有限公司 | Purification method of somalupeptide |
| CN113049690B (en) * | 2019-12-27 | 2022-07-22 | 翰宇药业（武汉）有限公司 | Polypeptide desalting method |
| US20230082544A1 (en) | 2020-02-18 | 2023-03-16 | Novo Nordisk A/S | Pharmaceutical formulations |
| WO2021224938A1 (en) * | 2020-05-05 | 2021-11-11 | Neuland Laboratories Limited | Improved process for the preparation of semaglutide |
| CN112175068B (en) * | 2020-09-28 | 2021-06-25 | 深圳深创生物药业有限公司 | Method for purifying semaglutide |
| CN112661815B (en) * | 2020-12-30 | 2022-12-27 | 江苏诺泰澳赛诺生物制药股份有限公司 | Method for purifying Tirzepatide |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101463080A (en) * | 2009-01-09 | 2009-06-24 | 深圳市翰宇药业有限公司 | Method for purifying nesiritide |
| CN101525382A (en) * | 2009-04-21 | 2009-09-09 | 深圳市翰宇药业有限公司 | Method of purifying pramlintide |
| CN102584982A (en) * | 2012-02-10 | 2012-07-18 | 深圳翰宇药业股份有限公司 | Method for purifying solid-phase synthetic coarse liraglutide |
| CN103122023A (en) * | 2013-03-08 | 2013-05-29 | 深圳翰宇药业股份有限公司 | Purification method for triptorelin |
| EP2651398A1 (en) * | 2010-12-16 | 2013-10-23 | Novo Nordisk A/S | Solid compositions comprising a glp-1 agonist and a salt of n-(8-(2-hydroxybenzoyl)amino)caprylic acid |
| CN104045705A (en) * | 2013-03-12 | 2014-09-17 | 深圳翰宇药业股份有限公司 | Synthetic method of liraglutide |
| CN104045707A (en) * | 2013-03-14 | 2014-09-17 | 深圳翰宇药业股份有限公司 | Purification method of teduglutide |
-
2014
- 2014-12-16 CN CN201410784130.9A patent/CN105777872B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101463080A (en) * | 2009-01-09 | 2009-06-24 | 深圳市翰宇药业有限公司 | Method for purifying nesiritide |
| CN101525382A (en) * | 2009-04-21 | 2009-09-09 | 深圳市翰宇药业有限公司 | Method of purifying pramlintide |
| EP2651398A1 (en) * | 2010-12-16 | 2013-10-23 | Novo Nordisk A/S | Solid compositions comprising a glp-1 agonist and a salt of n-(8-(2-hydroxybenzoyl)amino)caprylic acid |
| CN102584982A (en) * | 2012-02-10 | 2012-07-18 | 深圳翰宇药业股份有限公司 | Method for purifying solid-phase synthetic coarse liraglutide |
| CN103122023A (en) * | 2013-03-08 | 2013-05-29 | 深圳翰宇药业股份有限公司 | Purification method for triptorelin |
| CN104045705A (en) * | 2013-03-12 | 2014-09-17 | 深圳翰宇药业股份有限公司 | Synthetic method of liraglutide |
| CN104045707A (en) * | 2013-03-14 | 2014-09-17 | 深圳翰宇药业股份有限公司 | Purification method of teduglutide |
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