CN105777872A - Semaglutide purifying method - Google Patents
Semaglutide purifying method Download PDFInfo
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- CN105777872A CN105777872A CN201410784130.9A CN201410784130A CN105777872A CN 105777872 A CN105777872 A CN 105777872A CN 201410784130 A CN201410784130 A CN 201410784130A CN 105777872 A CN105777872 A CN 105777872A
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Abstract
The invention belongs to the technical field of pharmaceutical chemistry, and discloses a semaglutide purifying method. The purifying method comprises the following steps: taking a semaglutide sample to be purified, allowing the sample to go through a chemically bonded silica gel chromatographic column in order to carry out primary separation, carrying out gradient elution with an A1 solution and B solution mixed solution, and collecting an elution component to obtain a primary purification product; allowing the primary purification product to go through a chemically bonded silica gel chromatographic column in order to carry out secondary separation, carrying out gradient elution with an A2 solution and B solution mixed solution, and collecting an elution component to obtain a semaglutide solution, wherein the above A1 solution is a carbonate solution; the above A2 solution is a phosphate solution; and the B solution is an acetonitrile and isopropanol mixed solution with a volume ratio of acetonitrile to isopropanol of (8:2)-(9:1). The purifying method has the advantages of realization of high purity of the prepared semaglutide, substantial improvement of the semaglutide yield, simple operation, and facilitation of realization of large-scale preparation of semaglutide.
Description
Technical field
The invention belongs to field of pharmaceutical chemistry technology, particularly to the purification process of a kind of Sa Molu peptide.
Background technology
Diabetes are a kind of because of a series of clinical syndromes that internal insulin is absolute or relative deficiency causes, have very close associating with gene.The main clinical manifestation of diabetes is polydipsia, polyuria, polyphagia and weight loss, and containing glucose etc. in blood glucose height, urine.Diabetes are divided into four kinds of types: type Ⅰ diabetes mellitus, type Ⅱdiabetes mellitus, other types diabetes and gestational diabetes.Any kind of diabetes all can cause that the β cell in pancreas can not produce enough insulins to drop hypoglycemic concentration, causes the generation of hyperglycemia.World Health Organization's report of 2011 points out that the whole world has 3.46 hundred million people to suffer from diabetes, and estimation in 2004 has 3,400,000 people to die from the disease that hyperglycemia causes, and the Diabetes Death more than 80% occurs in low income and middle income country.
Type Ⅱdiabetes mellitus, it is once called as non-insulin-dependent diabetes mellitus (NIDDM) or Adult Onset's patients with type Ⅰ DM (adult-onsetdiabetes), it is a kind of metabolic disease, it is characterized by hyperglycemia, mainly relatively lacked by insulin resistant and insulin and cause.Wherein, patients with NIDDM accounts in diabetics about 90%, and all the other 10% are mainly type Ⅰ diabetes mellitus and gestational diabetes, so the research and development tool for the medicine of type Ⅱdiabetes mellitus is of great significance.
Antidiabetic drug kind for type Ⅱdiabetes mellitus is a lot, and the receptor agonism element of GLP-1 (GLP-1) is the focus of Recent study.Wherein, Sa Molu peptide (semaglutide) is one of receptor agonism element of GLP-1, and this medicine is developed by Novo Nordisk Co., Ltd of Denmark.Sa Molu peptide as one expendable subcutaneous injection formulation weekly, can play and good drop hypoglycemic effect, also have effect of fat-reducing simultaneously, and its chemistry is expressed as Nε26-{ 18-[N-(17-carboxyheptadecanoyl)-L-γ-glutamyl]-10-oxo-3,6,12,15-tetraoxa-9,18-diazaoctadecanoyl}-[8-(2-amino-2-propanoicacid), 34-L-arginine] humanglucagon-likepeptide1 (7-37), it has structure shown in Formulas I:
At present, the preparation method of Sa Molu peptide is mainly chemical synthesis.Owing to the peptide chain of Sa Molu peptide is longer, side chain contains in longer modification, peptide sequence and causes having in thick peptide isomer impurities containing, for example isomerized aminoacid easy in the building-up processes such as Ser, so the impurity produced in synthesis is more, it is necessary to the chemical synthesis gained thick peptide of Sa Molu peptide is carried out further purification.But, in prior art, the yield of Sa Molu peptide purification method is relatively low, causes that the purification of Sa Molu peptide becomes one of bottleneck of the difficult point in Sa Molu peptide preparation technology, Sa Molu peptide industrialization.It is therefore desirable to explore the purification process of a kind of Sa Molu peptide, to improve the yield of Sa Molu peptide.
Summary of the invention
In view of this, the goal of the invention of the present invention is in that to provide the purification process of a kind of Sa Molu peptide.The purification process of Sa Molu peptide provided by the invention is simple to operate, improves the yield of Sa Molu peptide, is more beneficial for the industrialized great production of Sa Molu peptide.
In order to realize the goal of the invention of the present invention, the present invention adopts the following technical scheme that:
The invention provides the purification process of a kind of Sa Molu peptide, comprising:
Take Sa Molu peptide sample to be purified, separate through chemical bonding silica gel chromatographic column first, carry out gradient elution with the mixed solution of A1 solution Yu B solution, collect elution fraction, obtain first time purified；
Take gained first time purified, separate through chemical bonding silica gel chromatographic column second, carry out gradient elution with the mixed solution of A2 solution Yu B solution, collect 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 wherein acetonitrile is (8:2)～(9:1) with the ratio of the volume of isopropanol.
The peptide chain of Sa Molu peptide is longer, branched structure is complicated, and the impurity produced in building-up process is many, so needing the Sa Molu peptide of synthesis is purified.In the present invention, by studying aminoacid stronger containing hydrophobicity in the peptide sequence having found that Sa Molu peptide, Sa Molu peptide dissolubility in aqueous is caused relatively low, difficult lower prop in purge process, cause damage, add purification difficulty, have impact on the yield of Sa Molu peptide.The present invention is by using chemical bonding silica gel as fixing phase, after loading, carrying out gradient elution with carbonate, it is possible to the most of isomer impurities in Sa Molu peptide to be purified and other are difficult to the magazins' layout, the removal that separate；Adopt phosphate solution as mobile phase afterwards, carry out gradient elution, remove difficulty except minute impurities and efficiently solve difficult lower prop, problem that yield is low.Experimental result confirms, purification process gained Sa Molu peptide purity provided by the invention is high, yield is high and easy and simple to handle, prepared by the scale being advantageously implemented Sa Molu peptide.
In some embodiments of the invention, in purification process provided by the invention, in B solution, the ratio of the volume of acetonitrile and isopropanol 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, the concentration of carbonate solution is 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 is 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 the other embodiment of the present invention, in purification process provided by the invention, with the pH value of Tetramethylammonium hydroxide adjustment A1 solution 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 the other embodiment of the present invention, in purification process provided by the invention, the concentration of phosphate 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 is pH2.0, pH2.5 or pH3.0.In the other embodiment of the present invention, in purification process provided by the invention, regulate pH to the pH2.0～pH3.0 of A2 solution with phosphoric acid.
Preferably, in purification process provided by the invention, in the first separation process, in mobile phase used by gradient elution, A1 solution is 62%:38% → 50%:50% with the Volume fraction of B solution.
Preferably, in purification process provided by the invention, in the second separation process, in mobile phase used by gradient elution, the Volume fraction of A2 solution and B solution is 60%:40% → 50%:50%.
Preferably, in purification process provided by the invention, in the first separation process, the carbonate in mobile phase used by gradient elution is the one in ammonium carbonate, ammonium hydrogen carbonate or potassium bicarbonate or both things mixed above.
Preferably, in purification process provided by the invention, in the second separation process, the phosphate solution in mobile phase used by gradient elution is the one in sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate or both things mixed above.
Preferably, in purification process provided by the invention, in the first separation process, collecting the Volume fraction of A1 solution and B solution in the mobile phase corresponding to elution fraction is 56%:44% → 52%:48%.
Preferably, in purification process provided by the invention, in the second separation process, collecting the Volume fraction of A2 solution and B solution in the mobile phase corresponding to elution fraction is 54%:46% → 51%:49%.
Preferably, in purification process provided by the invention, chemical bonding silica gel chromatographic column used in the first separation process is C8 silica gel chromatographic column or butyl bonded silica gel chromatographic column.In some embodiments of the invention, in purification process provided by the invention, 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, chemical bonding silica gel chromatographic column used in the second separation process is C8 silica gel chromatographic column or butyl bonded silica gel chromatographic column.In some embodiments of the invention, in purification process provided by the invention, 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, in chemical bonding silica gel chromatographic column used in the first separation process, the second separation process, chemical bonding silica gel chromatographic column used can be identical, it is also possible to different, both are independent of each other.In some embodiments of the invention, in purification process provided by the invention, chemical bonding silica gel used in chemical bonding silica gel chromatographic column used in the first separation process, the second separation process is identical.
In some embodiments of the invention, in purification process provided by the invention, the specification of chemical bonding silica gel chromatographic column used in the first separation process includes:
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, the specification of chemical bonding silica gel chromatographic column used in the second separation process includes:
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 the other embodiment of the present invention; in purification process provided by the invention; the synthetic method of Sa Molu peptide sample to be purified comprises the following steps: first synthesizing amino acid resin; progressively coupling synthesizes main chain peptide resin afterwards; then the protection base of Lys side chain is removed; the progressively aminoacid of coupling side chain and other group again, last cutting resin obtains the thick peptide of Sa Molu peptide, namely obtains Sa Molu peptide sample to be purified.
In some embodiments of the invention, in purification process provided by the invention, the first separation process takes and also includes before Sa Molu peptide sample to be purified carries out loading rinsing chemical bonding silica gel chromatographic column with acetonitrile solution.In some embodiments of the invention, in purification process provided by the invention, in the first separation process, when rinsing chemical bonding silica gel chromatographic column, in acetonitrile solution used, the percent by volume of acetonitrile is 50%～80%.
In the other embodiment of the present invention, in purification process provided by the invention, the second separation process takes first time purified and also includes before carrying out loading rinsing chemical bonding silica gel chromatographic column with acetonitrile solution.In the other embodiment of the present invention, in purification process provided by the invention, in the second separation process, when rinsing chemical bonding silica gel chromatographic column, in acetonitrile solution used, the percent by volume of acetonitrile is 50%～80%.
Preferably, in purification process provided by the invention, also including into the step of salt, this step specifically includes:
Take gained Sa Molu peptide solution, using chemical bonding silica gel chromatographic column as fixing phase, after loading, through becoming salt, eluting, collect elution fraction, get Sa Molu peptide salt；
Wherein, eluting is gradient elution, and the mobile phase used by eluting is the mixed solution of water and acetonitrile, and in this solution, the volume fraction of acetonitrile is gradually increased.
In some embodiments of the invention, in purification process provided by the invention, in salt-forming steps, chemical bonding silica gel chromatographic column is C8 silica gel chromatographic column or butyl bonded silica gel chromatographic column.In the other embodiment of the present invention, in purification process provided by the invention, 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, the specification of chemical bonding silica gel chromatographic column used in salt-forming steps includes:
5cm × 25cm (pillar diameter × length), 10cm × 25cm (pillar diameter × length), 15cm × 25cm (pillar diameter × length).
In the present invention, chemical bonding silica gel chromatographic column used in Sa Molu peptide salt-forming steps process, can be identical with chemical bonding silica gel chromatographic column used in chemical bonding silica gel chromatographic column used in the first separation process, the second separation process, it is also possible to different.
In the other embodiment of the present invention, in purification process provided by the invention, in salt-forming steps, become the mixed solution that solution is acetonitrile and ammonium acetate used by salt.In the other embodiment of the present invention, in purification process provided by the invention, in salt-forming steps, becoming in the mixed solution of the acetonitrile used by 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 the other embodiment of the present invention, in purification process provided by the invention, in salt-forming steps, the pH value becoming the mixed solution of the acetonitrile used by salt, ammonium acetate and water is pH6.5～pH7.0.
In the other embodiment of the present invention, in purification process provided by the invention, in salt-forming steps, before loading, also include the step rinsing chemical bonding silica gel chromatographic column with acetonitrile solution.In the other embodiment of the present invention, in purification process provided by the invention, in salt-forming steps, rinsing the percentage by volume of acetonitrile in acetonitrile solution used by chemical bonding silica gel chromatographic column before loading is 50%～80%.
In the other embodiment of the present invention, in purification process provided by the invention, in salt-forming steps, in the mobile phase used by eluting, water is 60%:40% → 40%:60% with the Volume fraction of acetonitrile.
In the other embodiment of the present invention, in purification process provided by the invention, in salt-forming steps, collecting the Volume fraction of water and acetonitrile in the mobile phase corresponding to elution fraction is 65%:35% → 45%:55%.
The invention provides the purification process of a kind of Sa Molu peptide.This purification process includes: takes Sa Molu peptide sample to be purified, separates through chemical bonding silica gel chromatographic column first, carries out gradient elution with the mixed solution of A1 solution Yu B solution, collects elution fraction, obtains first time purified；Take first time purified, separate through chemical bonding silica gel chromatographic column second, carry out gradient elution with the mixed solution of A2 solution Yu B solution, collect 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 of acetonitrile and the volume of isopropanol is (8:2)～(9:1).Experimental result confirms, the Sa Molu peptide purity that purification process provided by the invention prepares is high, significantly improves the yield of Sa Molu peptide, and simple to operate, prepared by the scale being advantageously implemented Sa Molu peptide.
Accompanying drawing explanation
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, and 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.
Detailed description of the invention
The invention discloses the purification process of a kind of Sa Molu peptide.Those skilled in the art are referred to present disclosure, implement the method, it is accordingly required in particular to it is noted that all similar replacements and change apparent to those skilled in the art, they are considered as including in the present invention.The purification process of the present invention is described already by preferred embodiment, and methods herein and application substantially can be modified or suitably change and combination by related personnel in without departing from present invention, spirit and scope, realize and apply the technology of the present invention.
Reagent used in the purification process of a kind of Sa Molu peptide provided by the invention and raw material all can be buied by market.
In order to make those skilled in the art better understood when technical scheme, below in conjunction with embodiment, the present invention is expanded on further:
The purification of the thick peptide of embodiment 1: Sa Molu peptide
Experiment material is originated: chromatographic column brand Kromasil；Ammonium hydrogen carbonate is purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Tetramethylammonium hydroxide is purchased from Aladdin；Sodium dihydrogen phosphate is purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Ammonia is purchased from Guangdong Guanghua 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 namely to be purified): company synthesizes voluntarily.
Synthetic method particularly as follows:
Adopting Fmoc-Gly-king's resin is initial resin; add in solid state reaction post; wash 2 times with DMF; after the swelling Fmoc-Gly-king's resin of DMF 30 minutes, remove Fmoc protection with DBLK, then wash 4 times with DMF; DCM washes 2 times; detecting color of resin with ninhydrin method, resin has color, represents that Fmoc removes.Weigh appropriate Fmoc-Arg (Pbf)-OH, HOBt (1.8mmol), PyBOP and be dissolved in DCM and the DMF mixed solution that volume ratio is 11, add under ice-water bath after DIPEA activates 3min and add in solid state reaction post, room temperature reaction 2 hours.Reaction end is judged with ninhydrin method detection, if resin water white transparency, then it represents that reacting completely, this criterion would judge reaction end with ninhydrin method detection suitable in subsequent content.
nullRepeat above-mentioned elimination Fmoc protection and add corresponding aminoacid and carry out the step of coupling，Peptide sequence according to Sa Molu peptide main chain，It 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、The coupling of Fmoc-Ala-OH and Boc-His (Trt)-OH.
Dichloromethane is solvent, adds appropriate phenyl silane, reacts 3 minutes, adds Pd (PPh3)4, room temperature reaction 45 minutes, to take out reactant liquor, detect color of resin with ninhydrin method, resin has color, represents that Alloc removes.
Weigh appropriate Fmoc-AEEA-OH, PyBOP, HOBt, dissolve with DMF, add DIPEA under ice-water bath and activate 3 minutes, add reaction column and react 2 hours, judge reaction end with ninhydrin method detection.Reaction terminates, and DBLK removes Fmoc, DMF and washs 6 times.Same method coupling Fmoc-AEEA-OH again, Fmoc-Glu-OtBu, octadecane diacid, reaction is shunk with methanol after terminating, and dried in vacuum overnight obtains peptide resin.
The peptide resin obtained, joins in reactor, prepares lytic reagent by the volume ratio of TFA thioanisole methyl phenyl ethers anisole EDT=90 523, is poured into by lytic reagent in peptide resin, room temperature reaction 2.5 hours.Reaction terminates, and filters resin, collects filtrate.Using TFA washing resin, merging filtrate, filtrate joined in absolute ether and precipitate, centrifugal, absolute ether washs, 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 that in Fig. 2, figure, peak T is Sa Molu peptide, calculates with external standard method, and wherein the content of Sa Molu peptide is 52%.
The mixed solution (volume ratio=15:5:80 of acetonitrile, ammonia and water) of thick for Sa Molu peptide peptide 2.0g acetonitrile, ammonia and water is dissolved, filters, collect filtrate standby.
1, the first step: first separates
Purification condition: chromatographic column: the chromatographic column with butyl bonded silica gel chromatographic column for fixing phase, pillar diameter and length is: 5cm × 25cm.Mobile phase: A1 phase: 50mmol/L ammonium bicarbonate soln solution, adjusts pH to 7.5 by Tetramethylammonium hydroxide；B phase: trifluoroacetic acid aqueous solution and isopropyl alcohol mixture (ratio of acetonitrile and the volume of isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient: B%:38% → 50%, 50-70min.
Purge process: rinse chromatographic column well rear loading with the acetonitrile solution of 50%, applied sample amount is the filtrate being made up of the 2g thick peptide of Sa Molu peptide.After loading, wash, linear gradient elution 60min.Collecting purpose peak, the elution fraction that purpose peak is corresponding is: B%:44% → 48%.By the peptide solution of collection in water temperature less than 32 DEG C when vacuum rotary steam be concentrated into about 15mg/mL, obtain first time purified, obtain altogether first time purified 0.82g (content meter with wherein peptide).
2, second step: second separates
Purification condition: the chromatographic column with butyl bonded silica gel chromatographic column for fixing phase, pillar diameter and length is: 5cm × 25cm.Mobile phase: A2 phase: 50mmol/L sodium dihydrogen phosphate ammonia adjusts pH to 2.0；B phase: trifluoroacetic acid aqueous solution and isopropyl alcohol mixture (ratio of acetonitrile and the volume of isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient: B%:40% → 50%, 50-70min.
Purge process: rinse chromatographic column well rear loading with the acetonitrile of 50%, applied sample amount is the first time purified 0.82g (content meter with wherein peptide) of first step gained.After loading, wash, linear gradient elution 60min.Collecting purpose peak, the elution fraction that purpose peak is corresponding is: B%:46% → 49%.By the peptide solution of collection in water temperature less than 32 DEG C when vacuum rotary steam be concentrated into about 15mg/mL, i.e. get Sa Molu peptide solution, obtain altogether the Sa Molu peptide solution containing 0.55g Sa Molu peptide.
3, turning salt: rinse butyl bonded silica gel chromatographic column well rear loading with the acetonitrile solution of 50%, applied sample amount is the Sa Molu peptide solution containing 0.55g Sa Molu peptide, rinses 30min with the Spirit of Mindererus. (pH is 6.5) of 0.1% containing 5% acetonitrile.Finally carry out gradient elution with water and acetonitrile, wherein, acetonitrile gradient: B%:40% → 60%, 40min, collect purpose peak, the elution fraction that purpose peak is corresponding is: B%:35% → 55%.The purpose peptide solution of collection vacuum rotary steam at water temperature is less than 32 DEG C is gone to suitable size cillin bottle after being concentrated into about 40mg/mL, carries out lyophilization afterwards, obtain white powdery solids 0.41g.
Gained white powdery solids is carried out Mass Spectrometer Method, and qualification result is shown in Fig. 3；Carrying out HPLC detection, testing result is shown in Fig. 4.It can be seen that the purity of Sa Molu peptide is 99.38% from HPLC chromatogram, single impurity is respectively less than 0.15%.Purification yield is 41% (computational methods are: quality × 100% of Sa Molu peptide in the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide)；Total recovery is 20.5%, (computational methods are: quality × 100% of the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide).
The thick peptide purification of embodiment 2: Sa Molu peptide
Experiment material is originated: chromatographic column brand Kromasil；Potassium bicarbonate is purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Tetramethylammonium hydroxide is purchased from Aladdin；Potassium dihydrogen phosphate is purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Ammonia is purchased from Guangdong Guanghua 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 namely to be purified): identical with embodiment 1, wherein the content of Sa Molu peptide is 52%.
The mixed solution (volume ratio=15:5:80 of acetonitrile, ammonia and water) of thick for Sa Molu peptide peptide 2.0g acetonitrile, water and ammonia is dissolved, filters, collect filtrate standby.
1, the first step: first separates
Purification condition: chromatographic column: the chromatographic column with butyl bonded silica gel for fixing phase, pillar diameter and length is: 5cm × 25cm.Mobile phase: A1 phase: 50mmol/L potassium bicarbonate solution solution, adjusts pH to 7.5 by Tetramethylammonium hydroxide；B phase: trifluoroacetic acid aqueous solution and isopropyl alcohol mixture (ratio of acetonitrile and the volume of isopropanol is 8:2).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient: B%:38% → 50%, 50-70min.
Purge process: rinse chromatographic column well rear loading with the acetonitrile solution of 80%, applied sample amount is the filtrate being made up of the 2g thick peptide of Sa Molu peptide.After loading, wash, linear gradient elution 60min.Collecting purpose peak, the elution fraction that purpose peak is corresponding is: B%:44% → 48%.By the peptide solution of collection in water temperature less than 32 DEG C when vacuum rotary steam be concentrated into about 15mg/mL, obtain first time purified, obtain altogether first time purified 0.88g (content meter with wherein peptide).
2, second step: second separates
Purification condition: chromatographic column: the chromatographic column with butyl bonded silica gel for fixing phase, pillar diameter and length is: 5cm × 25cm.Mobile phase: A2 phase: 20mmol/L potassium dihydrogen phosphate ammonia adjusts pH to 3.0；B phase: trifluoroacetic acid aqueous solution and isopropyl alcohol mixture (ratio of acetonitrile and the volume of isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient: B%:40% → 50%, 50-70min.
Purge process: rinse chromatographic column well rear loading with the acetonitrile of 80%, applied sample amount is the first time purified 0.88g (content meter with wherein peptide) of first step gained.After loading, wash, linear gradient elution 60min.Collecting purpose peak, the elution fraction that purpose peak is corresponding is: B%:46% → 49%.By the peptide solution of collection in water temperature less than 32 DEG C when vacuum rotary steam be concentrated into about 15mg/mL, i.e. get Sa Molu peptide solution, obtain altogether the Sa Molu peptide solution containing 0.5g Sa Molu peptide.
3, turning salt: rinse butyl bonded silica gel chromatographic column well rear loading with the acetonitrile solution of 80%, applied sample amount is the Sa Molu peptide solution containing 0.5g Sa Molu peptide.15min is rinsed with the Spirit of Mindererus. (pH is 7.0) of 0.4% 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 elution fraction that purpose peak is corresponding is: B%:35% → 55%.The purpose peptide solution of collection vacuum rotary steam at water temperature is less than 32 DEG C is gone to suitable size cillin bottle after being concentrated into about 45mg/mL, carries out lyophilization afterwards, obtain white powdery solids 0.4g.
Gained white powdery solids is carried out HPLC detection, and testing result is shown in Fig. 5, and its purity is 99.25%, and single impurity is respectively less than 0.15%.Purification yield is 40% (computational methods are: quality × 100% of Sa Molu peptide in the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide)；Total recovery is 20.2%, (computational methods are: quality × 100% of the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide).
The thick peptide purification of embodiment 3: Sa Molu peptide
Experiment material is originated: chromatographic column brand Kromasil；Ammonium carbonate is purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Tetramethylammonium hydroxide is purchased from Aladdin；Potassium dihydrogen phosphate is purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Ammonia is purchased from Guangdong Guanghua 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 namely to be purified): identical with embodiment 1, wherein the content of Sa Molu peptide is 52%.
Thick for Sa Molu peptide peptide 2.0g is dissolved, filters, collect filtrate standby.
1, the first step: first separates
Purification condition: chromatographic column: the chromatographic column with butyl bonded silica gel for fixing phase, pillar diameter and length is: 5cm × 25cm.Mobile phase: A1 phase: 20mmol/L sal volatile, adjusts pH to 8.5 by Tetramethylammonium hydroxide；B phase: trifluoroacetic acid aqueous solution and isopropyl alcohol mixture (ratio of acetonitrile and the volume of isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient: B%:38% → 50%, 50-70min.
Purge process: rinse chromatographic column well rear loading with the acetonitrile solution of 75%, applied sample amount is the filtrate being made up of the 2g thick peptide of Sa Molu peptide.After loading, wash, linear gradient elution 60min.Collecting purpose peak, the elution fraction that purpose peak is corresponding is: B%:44% → 48%.By the peptide solution of collection in water temperature less than 32 DEG C when vacuum rotary steam be concentrated into about 15mg/mL, obtain first time purified, obtain altogether first time purified 0.75g (content meter with wherein peptide).
2, second step: second separates
Purification condition: chromatographic column: the chromatographic column with butyl bonded silica gel for fixing phase, pillar diameter and length is: 5cm × 25cm.Mobile phase: A2 phase: 50mmol/L potassium dihydrogen phosphate ammonia adjusts pH to 2.0；B phase: trifluoroacetic acid aqueous solution and isopropyl alcohol mixture (ratio of acetonitrile and the volume of isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient: B%:40% → 50%, 50-70min.
Purge process: rinse chromatographic column well rear loading with the acetonitrile solution of 75%, applied sample amount is the first time purified 0.75g (content meter with wherein peptide) of first step gained.After loading, wash, linear gradient elution 60min.Collecting purpose peak, the elution fraction that purpose peak is corresponding is: B%:46% → 49%.By the peptide solution of collection in water temperature less than 32 DEG C when vacuum rotary steam be concentrated into about 15mg/mL, i.e. get Sa Molu peptide solution, obtain altogether the Sa Molu peptide solution containing 0.62g Sa Molu peptide.Carry out turning salt.
3, turning salt: rinse butyl bonded silica gel chromatographic column well rear loading with the acetonitrile solution of 75%, applied sample amount is the Sa Molu peptide solution containing 0.62g Sa Molu peptide.20min is rinsed with the Spirit of Mindererus. (pH is 6.8) of 0.3% 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 elution fraction that purpose peak is corresponding is: B%:35% → 55%.The purpose peptide solution of collection vacuum rotary steam at water temperature is less than 32 DEG C is gone to suitable size cillin bottle after being concentrated into about 45mg/mL, carries out lyophilization afterwards, obtain white powdery solids 0.5g.
Gained white powdery solids is carried out HPLC detection, and testing result is shown in Fig. 6, and its purity is 99.38%, and single impurity is respectively less than 0.15%.Purification yield is 50% (computational methods are: quality × 100% of Sa Molu peptide in the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide)；Total recovery is 25%, (computational methods are: quality × 100% of the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide).
The thick peptide purification of embodiment 4: Sa Molu peptide
Experiment material is originated: chromatographic column brand Kromasil；Ammonium hydrogen carbonate is purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Tetramethylammonium hydroxide is purchased from Aladdin；Sodium dihydrogen phosphate is purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Ammonia is purchased from Guangdong Guanghua 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 namely to be purified): identical with embodiment 1, wherein the content of Sa Molu peptide is 52%.
The mixed solution (volume ratio=15:5:80 of acetonitrile, ammonia and water) of thick for Sa Molu peptide peptide 2.0g acetonitrile, water and ammonia is dissolved, filters, collect filtrate standby.
1, the first step: first separates
Purification condition: chromatographic column: the chromatographic column with butyl bonded silica gel for fixing phase, pillar diameter and length is: 5cm × 25cm.Mobile phase: A1 phase: 50mmol/L ammonium bicarbonate soln, adjusts pH to 8.5 by Tetramethylammonium hydroxide；B phase: trifluoroacetic acid aqueous solution and isopropyl alcohol mixture (ratio of acetonitrile and the volume of isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient: B%:38% → 50%, 50-70min.
Purge process: rinse chromatographic column well rear loading with the acetonitrile solution of 75%, applied sample amount is the filtrate being made up of the 2g thick peptide of Sa Molu peptide.After loading, wash, linear gradient elution 60min.Collecting purpose peak, the elution fraction that purpose peak is corresponding is: B%:44% → 48%.By the peptide solution of collection in water temperature less than 32 DEG C when vacuum rotary steam be concentrated into about 15mg/mL, obtain first time purified, obtain altogether first time purified 0.78g (content meter with wherein peptide).
Second step: second separates
Purification condition: chromatographic column: the chromatographic column with butyl bonded silica gel for fixing phase, pillar diameter and length is: 5cm × 25cm.Mobile phase: A2 phase: 50mmol/L sodium dihydrogen phosphate ammonia adjusts pH to 3.0；B phase: trifluoroacetic acid aqueous solution and isopropyl alcohol mixture (ratio of acetonitrile and the volume of isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient: B%:40% → 50%, 50-70min.
Purge process: rinse chromatographic column well rear loading with the acetonitrile solution of 75%, applied sample amount is the first time purified 0.78g (content meter with wherein peptide) of first step gained.After loading, wash, linear gradient elution 60min.Collecting purpose peak, the elution fraction that purpose peak is corresponding is: B%:46% → 49%.By the peptide solution of collection in water temperature less than 32 DEG C when vacuum rotary steam be concentrated into about 15mg/mL, i.e. get Sa Molu peptide solution, obtain altogether the Sa Molu peptide solution containing 0.6g Sa Molu peptide, carry out turning salt.
3, turning salt: rinse butyl bonded silica gel chromatographic column well rear loading with the acetonitrile solution of 75%, applied sample amount is the Sa Molu peptide solution containing 0.6g Sa Molu peptide, rinses 20min with the Spirit of Mindererus. (pH is 6.5) of 0.2% 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 elution fraction that purpose peak is corresponding is: B%:35% → 55%.The purpose peptide solution of collection vacuum rotary steam at water temperature is less than 32 DEG C is gone to suitable size cillin bottle after being concentrated into about 15mg/mL, carries out lyophilization afterwards, obtain white powdery solids 0.45g.
Gained white powdery solids is carried out HPLC detection, and testing result is shown in Fig. 7, and its purity is 99.30%, and single impurity is respectively less than 0.15%.Purification yield is 45% (computational methods are: quality × 100% of Sa Molu peptide in the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide)；Total recovery is 22.5%, (computational methods are: quality × 100% of the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide).
The thick peptide purification of embodiment 5: Sa Molu peptide
Experiment material is originated: chromatographic column brand Kromasil；Ammonium hydrogen carbonate is purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Tetramethylammonium hydroxide is purchased from Aladdin；Potassium dihydrogen phosphate is purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Ammonia is purchased from Guangdong Guanghua 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 namely to be purified): identical with embodiment 1, wherein the content of Sa Molu peptide is 52%.
The mixed solution (volume ratio=15:5:80 of acetonitrile, ammonia and water) of thick for Sa Molu peptide peptide 2.0g acetonitrile, water and ammonia is dissolved, filters, collect filtrate standby.
1, the first step: first separates
Purification condition: chromatographic column: the chromatographic column with butyl bonded silica gel for fixing phase, pillar diameter and length is: 5cm × 25cm.Mobile phase: A1 phase: 150mmol/L ammonium bicarbonate soln solution, adjusts pH to 8.0 by Tetramethylammonium hydroxide；B phase: trifluoroacetic acid aqueous solution and isopropyl alcohol mixture (ratio of acetonitrile and the volume of isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient: B%:38% → 50%, 50-70min.
Rinsing chromatographic column well rear loading with the acetonitrile solution of 75%, applied sample amount is the filtrate being made up of the 2g thick peptide of Sa Molu peptide.After loading, wash, linear gradient elution 60min.Collecting purpose peak, the elution fraction that purpose peak is corresponding is: B%:44% → 48%.By the peptide solution of collection in water temperature less than 32 DEG C when vacuum rotary steam be concentrated into about 15mg/mL, obtain first time purified, obtain altogether first time purified 0.8g (content meter with wherein peptide).
2, second step: second separates
Purification condition: chromatographic column: the chromatographic column with butyl bonded silica gel for fixing phase, pillar diameter and length is: 5cm × 25cm.Mobile phase: A2 phase: 100mmol/L dipotassium hydrogen phosphate solution ammonia adjusts pH to 2.5；B phase: the mixed solution of trifluoroacetic acid aqueous solution and isopropanol (ratio of ethanol and the volume of isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient: B%:40% → 50%, 50-70min.
Purge process: rinse chromatographic column well rear loading with the acetonitrile solution of 50%, applied sample amount is the first time purified 0.8g (content meter with wherein peptide) of first step gained.After loading, wash, linear gradient elution 60min.Collecting purpose peak, the elution fraction that purpose peak is corresponding is: B%:46% → 49%.By the peptide solution of collection in water temperature less than 32 DEG C when vacuum rotary steam be concentrated into about 15mg/mL, i.e. get Sa Molu peptide solution, obtain altogether the Sa Molu peptide solution containing 0.57g Sa Molu peptide, carry out turning salt.
3, turning salt: rinse butyl bonded silica gel chromatographic column well rear loading with the acetonitrile solution of 50%, applied sample amount is the Sa Molu peptide solution containing 0.57g Sa Molu peptide, rinses 30min with the Spirit of Mindererus. (pH is 6.5) of 0.4% containing 5% acetonitrile.Last gradient elution, carries out gradient elution with water and acetonitrile, wherein, acetonitrile gradient: B%:40% → 60%, 40min, collect purpose peak, the elution fraction that purpose peak is corresponding is: B%:35% → 55%.The purpose peptide solution of collection vacuum rotary steam at water temperature is less than 32 DEG C is gone to suitable size cillin bottle after being concentrated into about 15mg/mL, carries out lyophilization afterwards, obtain white powdery solids 0.5g.
Gained white powdery solids is carried out HPLC detection, and testing result is shown in Fig. 8, and its purity is 99.20%, and single impurity is respectively less than 0.15%.Purification yield is 50% (computational methods are: quality × 100% of Sa Molu peptide in the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide)；Total recovery is 25%, (computational methods are: quality × 100% of the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide).
The thick peptide purification of embodiment 6: Sa Molu peptide
Experiment material is originated: chromatographic column brand Kromasil；Ammonium hydrogen carbonate is purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Tetramethylammonium hydroxide is purchased from Aladdin；Sodium dihydrogen phosphate is purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Ammonia is purchased from Guangdong Guanghua 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 namely to be purified): identical with embodiment 1, wherein the content of Sa Molu peptide is 52%.
The mixed solution (volume ratio=15:5:80 of acetonitrile, ammonia and water) of thick for Sa Molu peptide peptide 15g acetonitrile, water and ammonia is dissolved, filters, collect filtrate standby.
1, the first step: first separates
Purification condition: chromatographic column: the chromatographic column with butyl bonded silica gel for fixing phase, pillar diameter and length is: 10cm × 25cm.Mobile phase: A1 phase: 100mmol/L ammonium bicarbonate soln solution, adjusts pH to 8.0 by Tetramethylammonium hydroxide；B phase: the mixed solution of trifluoroacetic acid aqueous solution and isopropanol (ratio of acetonitrile and the volume of isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient: B%:38% → 50%, 50-70min.
Purge process: rinse chromatographic column well rear loading with the acetonitrile solution of 75%, applied sample amount is the filtrate being made up of the 15g thick peptide of Sa Molu peptide.After loading, wash, linear gradient elution 60min.Collecting purpose peak, the elution fraction that purpose peak is corresponding is: B%:44% → 48%.By the peptide solution of collection in water temperature less than 32 DEG C when vacuum rotary steam be concentrated into about 15mg/mL, obtain first time purified, obtain altogether first time purified 6g (content meter with wherein peptide).
2, second step: second separates
Purification condition: chromatographic column: the chromatographic column with butyl bonded silica gel for fixing phase, pillar diameter and length is: 10cm × 25cm.Mobile phase: A2 phase: 100mmol/L sodium dihydrogen phosphate, adjusts pH to 2.5 with ammonia；B phase: the mixed solution of trifluoroacetic acid aqueous solution and isopropanol (ratio of acetonitrile and the volume of isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient: B%:40% → 50%, 50-70min.
Purge process: rinse chromatographic column well rear loading with the acetonitrile of 80%, applied sample amount is first step gained first time purified 6g (content meter with wherein peptide).After loading, wash, linear gradient elution 60min.Collecting purpose peak, the elution fraction that purpose peak is corresponding is: B%:46% → 49%.By the peptide solution of collection in water temperature less than 32 DEG C when vacuum rotary steam be concentrated into about 15mg/mL, i.e. get Sa Molu peptide solution, obtain altogether the Sa Molu peptide solution containing 4.8g Sa Molu peptide, carry out turning salt.
3, salt is turned: rinse butyl bonded silica gel chromatographic column well rear loading with the acetonitrile solution of 80%, applied sample amount is that the Sa Molu peptide containing 4.8g Sa Molu peptide is molten, 15min is rinsed with the Spirit of Mindererus. (pH is 7.0) of 0.4% containing 5% acetonitrile, finally with gradient elution, carry out gradient elution with water and acetonitrile, wherein, acetonitrile gradient: B%:40% → 60%, 40min, collects purpose peak, and the elution fraction that purpose peak is corresponding is: B%:35% → 55%.The purpose peptide solution of collection vacuum rotary steam at water temperature is less than 32 DEG C is gone to suitable size cillin bottle after being concentrated into about 45mg/mL.Carry out lyophilization afterwards, obtain white powdery solids 4.2g.
Gained white powdery solids is carried out HPLC detection, and testing result is shown in Fig. 9, and its purity is 99.38%, and single impurity is respectively less than 0.15%.Purification yield is 53.8% (computational methods are: quality × 100% of Sa Molu peptide in the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide)；Total recovery is 28%, (computational methods are: quality × 100% of the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide).
The thick peptide purification of embodiment 7: Sa Molu peptide
Experiment material is originated: chromatographic column brand Kromasil；Ammonium hydrogen carbonate is purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Tetramethylammonium hydroxide is purchased from Aladdin；Potassium dihydrogen phosphate is purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Ammonia is purchased from Guangdong Guanghua 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 namely to be purified): identical with embodiment 1, wherein the content of Sa Molu peptide is 52%.
Thick for Sa Molu peptide peptide 25g acetonitrile, water and ammonia (volume ratio=15:5:80 of acetonitrile, ammonia and water) are dissolved, filters, collect filtrate standby.
1, the first step: first separates
Purification condition: chromatographic column: the chromatographic column with butyl bonded silica gel for fixing phase, pillar diameter and length is: 15cm × 25cm.Mobile phase: A1 phase: 100mmol/L ammonium bicarbonate soln solution, adjusts pH to 8.0 by Tetramethylammonium hydroxide；B phase: trifluoroacetic acid aqueous solution and isopropyl alcohol mixture (ratio of ethanol and the volume of isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient: B%:38% → 50%, 50-70min.
Purge process: rinse chromatographic column well rear loading with the acetonitrile solution of 75%, applied sample amount is the filtrate being made up of the 25g thick peptide of Sa Molu peptide.After loading, wash, linear gradient elution 60min.Collecting purpose peak, the elution fraction that purpose peak is corresponding is: B%:44% → 48%.By the peptide solution of collection in water temperature less than 32 DEG C when vacuum rotary steam be concentrated into about 15mg/mL, obtain first time purified, obtain altogether first time purified 10g (content meter with wherein peptide).
2, second step: second separates
Purification condition: chromatographic column: the chromatographic column with butyl bonded silica gel for fixing phase, pillar diameter and length is: 15cm × 25cm.Mobile phase: A2 phase: 150mmol/L potassium dihydrogen phosphate ammonia adjusts pH to 2.5；B phase: trifluoroacetic acid aqueous solution and isopropyl alcohol mixture (ratio of acetonitrile and the volume of isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient: B%:40% → 50%, 50-70min.Sample size is 20-30g.
Purge process: rinse chromatographic column well rear loading with the acetonitrile of 60%, applied sample amount is first step gained first time purified 10g (content meter with wherein peptide).After loading, wash, linear gradient elution 60min.Collecting purpose peak, the elution fraction that purpose peak is corresponding is: B%:46% → 49%.By the peptide solution of collection in water temperature less than 32 DEG C when vacuum rotary steam be concentrated into about 15mg/mL, i.e. get Sa Molu peptide solution, obtain altogether the Sa Molu peptide solution containing 8.1g Sa Molu peptide, carry out turning salt.
3, salt is turned: rinse butyl bonded silica gel chromatographic column well rear loading with the acetonitrile solution of 60%, applied sample amount is the Sa Molu peptide solution containing 8.1g Sa Molu peptide, 30min is rinsed with the Spirit of Mindererus. (pH is 6.8) of 0.4% containing 5% acetonitrile, finally with gradient elution, carry out gradient elution with water and acetonitrile, wherein, acetonitrile gradient: B%:40% → 60%, 40min, collects purpose peak, and the elution fraction that purpose peak is corresponding is: B%:35% → 55%.By the purpose peptide solution of collection in water temperature less than 32 DEG C when vacuum rotary steam be concentrated into about 40mg/mL after go to suitable size cillin bottle.Carry out lyophilization afterwards, obtain white powdery solids 7.4g.
Gained white powdery solids is carried out HPLC detection, and testing result is shown in Figure 10, and its purity is 99.45%, and single impurity is respectively less than 0.15%.Purification yield is 56% (computational methods are: quality × 100% of Sa Molu peptide in the weight of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide)；Total recovery is 29.6%, (computational methods are: quality × 100% of the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide).
The thick peptide purification of embodiment 8: Sa Molu peptide
Experiment material is originated: chromatographic column brand Kromasil；Ammonium hydrogen carbonate is purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Tetramethylammonium hydroxide is purchased from Aladdin；Sodium dihydrogen phosphate is purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Ammonia is purchased from Guangdong Guanghua 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 namely to be purified): identical with embodiment 1, wherein the content of Sa Molu peptide is 52%.
Thick for Sa Molu peptide peptide 15g acetonitrile, water and ammonia (volume ratio=15:5:80 of acetonitrile, ammonia and water) are dissolved, filters, collect filtrate standby.
1, the first step: first separates
Purification condition: chromatographic column: the chromatographic column with C8 silica gel chromatographic column for fixing phase, pillar diameter and length is: 10cm × 25cm.Mobile phase: A1 phase: 100mmol/L ammonium bicarbonate soln solution, adjusts pH to 8.0 by Tetramethylammonium hydroxide；B phase: the mixed solution of trifluoroacetic acid aqueous solution and isopropanol (ratio of acetonitrile and the volume of isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient: B%:38% → 50%, 50-70min.
Purge process: rinse chromatographic column well rear loading with the acetonitrile solution of 75%, applied sample amount is the filtrate being made up of the 15g thick peptide of Sa Molu peptide.After loading, wash, linear gradient elution 60min.Collecting purpose peak, the elution fraction that purpose peak is corresponding is: B%:44% → 48%.By the peptide solution of collection in water temperature less than 32 DEG C when vacuum rotary steam be concentrated into about 15mg/mL, obtain first time purified, obtain altogether first time purified 5g (content meter with wherein peptide).
2, second step: second separates
Purification condition: chromatographic column: the chromatographic column with C8 silica gel chromatographic column for fixing phase, pillar diameter and length is: 10cm × 25cm.Mobile phase: A2 phase: 20mmol/L sodium dihydrogen phosphate, adjusts pH to 2.5 with ammonia；B phase: the mixed solution of trifluoroacetic acid aqueous solution and isopropanol (ratio of acetonitrile and the volume of isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient: B%:40% → 50%, 50-70min.
Purge process: rinse chromatographic column well rear loading with the acetonitrile of 80%, applied sample amount is by first step gained first time purified 5g (content meter with wherein peptide).After loading, wash, linear gradient elution 60min.Finally, carrying out eluting, linear gradient elution 60min, collect purpose peak, the elution fraction that purpose peak is corresponding is: B%:46% → 49%.By the peptide solution of collection in water temperature less than 32 DEG C when vacuum rotary steam be concentrated into about 15mg/mL, i.e. get Sa Molu peptide solution, obtain altogether the Sa Molu peptide solution containing 3.2g Sa Molu peptide, standby.
3, salt is turned: rinse C8 silica gel chromatographic column post well rear loading with the acetonitrile solution of 80%, applied sample amount is the Sa Molu peptide solution containing 3.2g Sa Molu peptide, 15min is rinsed with the Spirit of Mindererus. (pH is 7.0) of 0.4% containing 5% acetonitrile, finally with gradient elution, carry out gradient elution with water and acetonitrile, wherein, acetonitrile gradient: B%:40% → 60%, 40min, collects purpose peak, and the elution fraction that purpose peak is corresponding is: B%:35% → 55%.The purpose peptide solution of collection vacuum rotary steam at water temperature is less than 32 DEG C is gone to suitable size cillin bottle after being concentrated into about 45mg/mL.Carry out lyophilization afterwards, obtain white powdery solids 2.3g.
Gained white powdery solids is carried out HPLC detection, and testing result is shown in Figure 11, and its purity is 99.28%%, and single impurity is respectively less than 0.15%.Purification yield is 29% (computational methods are: quality × 100% of Sa Molu peptide in the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide)；Total recovery is 15.3%, (computational methods are: quality × 100% of the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide).
Comparative example
Experiment material is originated: chromatographic column brand Kromasil；Ammonium hydrogen carbonate is purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Tetramethylammonium hydroxide is purchased from Aladdin；Sodium dihydrogen phosphate is purchased from Chemical Reagent Co., Ltd., Sinopharm Group；Ammonia is purchased from Guangdong Guanghua 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 namely to be purified): identical with embodiment 1, wherein the content of Sa Molu peptide is 52%.
Thick for Sa Molu peptide peptide 2.0g acetonitrile, water and ammonia (volume ratio=15:5:80 of acetonitrile, ammonia and water) are dissolved, filters, collect filtrate standby.
1, the first step: first separates
Purification condition: chromatographic column: the chromatographic column with octadecyl silane for fixing phase, pillar diameter and length is: 5cm × 25cm.Mobile phase: A1 phase: 150mmol/L ammonium bicarbonate soln solution, adjusts pH to 8.0 by Tetramethylammonium hydroxide；B phase: trifluoroacetic acid aqueous solution and isopropyl alcohol mixture (ratio of acetonitrile and the volume of isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient: B%:40% → 55%, 50-70min.
Purge process: rinse chromatographic column well rear loading with the acetonitrile solution of 75%, applied sample amount is the filtrate being made up of the 2g thick peptide of Sa Molu peptide.After loading, wash, linear gradient elution 60min.Collecting purpose peak, the elution fraction that purpose peak is corresponding is: B%:44% → 48%.By the peptide solution of collection in water temperature less than 32 DEG C when vacuum rotary steam be concentrated into about 15mg/mL, obtain first time purified, obtain altogether first time purified 0.5g (content meter with wherein peptide).
2, second step: second separates
Purification condition: chromatographic column: the chromatographic column with octadecyl silane for fixing phase, pillar diameter and length is: 5cm × 25cm.Mobile phase: A2 phase: 100mmol/L sodium dihydrogen phosphate ammonia adjusts pH to 2.5；B phase: the mixed solution of trifluoroacetic acid aqueous solution and isopropanol (ratio of ethanol and the volume of isopropanol is 9:1).Flow velocity: 60-80mL/min.Detection wavelength: 230nm.Gradient: B%:42% → 52%, 50-70min.
Purge process: rinse chromatographic column well rear loading with the acetonitrile solution of 50%, applied sample amount is 0.5g first time purified (content meter with peptide therein) that the first step obtains.After loading, wash, linear gradient elution 60min.Collecting purpose peak, the elution fraction that purpose peak is corresponding is: B%:46% → 49%.By the peptide solution of collection in water temperature less than 32 DEG C when vacuum rotary steam be concentrated into about 15mg/mL, i.e. get Sa Molu peptide solution, obtain altogether the Sa Molu peptide solution containing 0.4g Sa Molu peptide, carry out turning salt.
3, turning salt: rinse butyl bonded silica gel chromatographic column well rear loading with the acetonitrile solution of 50%, applied sample amount is the Sa Molu peptide solution containing 0.4g Sa Molu peptide, rinses 30min with the Spirit of Mindererus. (pH is 6.5) of 0.4% containing 5% acetonitrile.Last gradient elution, gradient elution is carried out with water and acetonitrile, wherein, acetonitrile gradient: B%:40% → 60%, 40min, collects purpose peak, the purpose peptide solution of collection vacuum rotary steam at water temperature is less than 32 DEG C is gone to after being concentrated into about 15mg/mL suitable size cillin bottle, carry out lyophilization afterwards, obtain white powdery solids 0.2g.
Gained white powdery solids is carried out HPLC detection, and testing result is shown in Figure 12, and its purity is 98.2%, and single impurity is respectively less than 0.3%.Purification yield is 20% (computational methods are: quality × 100% of Sa Molu peptide in the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide)；Total recovery is 10%, (computational methods are: quality × 100% of the quality of gained Sa Molu peptide salt/thick peptide of Sa Molu peptide).
Below being only the preferred embodiment of the present invention, it is noted that above-mentioned preferred implementation is not construed as limitation of the present invention, protection scope of the present invention should be as the criterion with claim limited range.For those skilled in the art, without departing from the spirit and scope of the present invention, it is also possible to make some improvements and modifications, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (10)
1. the purification process of Yi Zhong Sa Molu peptide, it is characterised in that comprising:
Take Sa Molu peptide sample to be purified, separate through chemical bonding silica gel chromatographic column first, carry out gradient elution with the mixed solution of A1 solution Yu B solution, collect elution fraction, obtain first time purified；
Take described first time purified, separate through chemical bonding silica gel chromatographic column second, carry out gradient elution with the mixed solution of A2 solution Yu B solution, collect elution fraction, get Sa Molu peptide solution；
Described A1 solution is carbonate solution；
Described A2 solution is phosphate solution；
Described B solution is the mixed solution of acetonitrile and isopropanol, and the ratio of described acetonitrile and the volume of isopropanol is (8:2)～(9:1).
2. purification process according to claim 1, it is characterised in that the concentration of described carbonate solution is 20mmol/L～150mmol/L.
3. purification process according to claim 1, it is characterised in that the pH value of described A1 solution is 7.5～8.5.
4. purification process according to claim 1, it is characterised in that the concentration of described phosphate solution is 20mmol/L～150mmol/L.
5. purification process according to claim 1, it is characterised in that the pH value of described A2 solution is 2.0～3.0.
6. purification process according to claim 1, it is characterised in that in described first separation, in mobile phase used by described gradient elution, the Volume fraction of described A1 solution and described B solution is 62%:38% → 50%:50%.
7. purification process according to claim 1, it is characterised in that in described second separation, in mobile phase used by described gradient elution, the Volume fraction of described A2 solution and described B solution is 60%:40% → 50%:50%.
8. purification process according to claim 1, it is characterised in that in described second separation, the Volume fraction of A2 solution described in the mobile phase corresponding to described collection elution fraction and described B solution is 54%:46% → 51%:49%.
9. purification process according to claim 1, it is characterised in that in described second separation, described chemical bonding silica gel chromatographic column is C8 silica gel chromatographic column or butyl bonded silica gel chromatographic column.
10. purification process according to any one of claim 1 to 9, it is characterised in that also include into the step of salt, itself particularly as follows:
Take described Sa Molu peptide solution, using chemical bonding silica gel chromatographic column as fixing phase, after loading, through becoming salt, eluting, collect elution fraction, get Sa Molu peptide salt；
Described eluting is gradient elution, and the mobile phase used by described eluting is the mixed solution of water and acetonitrile, and the volume fraction of described acetonitrile is gradually increased.
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