CN109354622A - A kind of Suo Malu peptide purification filler special and its purification process - Google Patents
A kind of Suo Malu peptide purification filler special and its purification process Download PDFInfo
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- CN109354622A CN109354622A CN201811482820.3A CN201811482820A CN109354622A CN 109354622 A CN109354622 A CN 109354622A CN 201811482820 A CN201811482820 A CN 201811482820A CN 109354622 A CN109354622 A CN 109354622A
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- Prior art keywords
- peptide
- suo malu
- malu peptide
- purification
- phase
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/605—Glucagons
Abstract
The present invention relates to a kind of Suo Malu peptide purification filler special and its purification process, specific steps are as follows: the thick peptide of Suo Malu peptide is dissolved in acetonitrile solution and obtains thick peptide solution；Filtering is mixed in a certain proportion with reverse phase and ion-exchange packing as stationary phase, after filtrate loading, the mobile phase that mixes with organic phase and salt while carrying out HPLC linear gradient elution, fraction of the collection containing Suo Malu peptide；Take the third fraction through vacuum rotary steam concentration, be freeze-dried to get.90% or more can be purified to for one step of Suo Malu peptide of purity 30% or so, and applied sample amount is high, industrial production amplification is simple, and purification cycle is short, greatly reduces the production cost of enterprise.
Description
Technical field
The present invention relates to packing technique fields, are a kind of Suo Malu peptide purification filler special and its purifying side specifically
Method.
Background technique
Suo Malu peptide (Semaglutide) is a kind of new long-acting GLP-1 receptor agonists, not only has significant drop blood
Sugared curative effect also has apparent effect of weight reducing, which is also to have both hypoglycemic and slimming exercise for the 2nd kind in Novo Nordisk diabetes pipeline
The GLP-1 antidiabetic drug of effect (another kind is Liraglutide).The molecular formula of Suo Malu peptide be C187H291N45O59, corresponding point
Son amount is 4111.1, structurally, Suo Malu peptide needs 26 Lys connecting AEEA, glutamic acid and octadecanoid acid aliphatic chain,
Wherein, 8 use unnatural amino acid aminoisobutyric acid.Compared with Liraglutide, the aliphatic chain of Suo Malu peptide is longer, hydrophobicity
Increase, but Suo Malu peptide is modified through the AEEA of too short chain, hydrophily greatly enhances.It not only can be with albumin after AEEA modification
It combines closely, covers DPP-4 enzyme hydrolysis site, moreover it is possible to reduce renal excretion, biological half-life can be extended, reach macrocyclic effect
Fruit.
Peptide sequence structure is as follows:
H-His7-Ala8-Glu9-Gly10-Thr11-Phe12-Thr13-Ser14-Asp15-Val16-Ser17-
Ser18-Tyr19-Leu20-Glu21-Gly22-Gln23-Ala24-Ala25-Lys26(AEEA-AEEA--Glu-
OctadecanedioicAcidMono-tert-butylester)-Glu27-Phe28-Ile29-Ala30-Trp31-Leu32-
Val33-Arg34-Gly35-Arg36-Gly37-OH。
The solid-liquid synthetic method of traditional Suo Malu peptide, intermediate GLP-I (7-37)-OH are required to reversed-phase HPLC purifying, then
It is reacted under liquid-phase condition with Na-alkanoyl-Glu (ONSu)-OtBu, and since GLP-I (7-37)-OH N-terminal is unprotected
And Side chain protective group is totally removed, and will lead to and generates many impurity, it is difficult to it purifies, it is cumbersome.In order to guarantee Suo Malu peptide
Subsequent research and development and the quality of production need to continuously improve its production technology.Therefore, the Suo Malu of higher purity how is obtained
Peptide seems especially urgent for medicine manufacture.
For the existing purification process of Suo Malu peptide there are cumbersome, the period is long, and waste liquid is more, is unfavorable for environmental protection, with high costs
And the shortcomings that being unfavorable for large-scale production.In field of polypeptide purification, purity, maximum of the different purification strategies to final products
Single contaminant content and yield have a significant impact, and how under the premise of guaranteeing purity and yield, reduce purifying work as far as possible
The complexity of skill, maximum single contaminant and shortening synthesis cycle, are the research emphasis of technical staff instantly.
Summary of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of Suo Malu peptide purification filler specials and its pure
Change method.
The purpose of the present invention is achieved through the following technical solutions:
A kind of Suo Malu peptide purification filler special and its purification process
Step 1: the thick peptide of Suo Malu peptide taken, which is dissolved in acetonitrile solution, obtains thick peptide solution, filtering；
Step 2: the thick peptide solution is taken, stationary phase is mixed into reverse phase filler and ion-exchange packing, after filtrate loading,
The mobile phase that mixes with organic phase and salt while HPLC linear gradient elution is carried out, collects the fraction containing Suo Malu peptide；
Step 3: taking the third fraction through vacuum rotary steam concentration, be freeze-dried to get high-purity Suo Malu peptide.
The present invention is a kind of Suo Malu peptide purification filler special and its purification process, and further preferred technical solution is such as
Under:
(1) the thick peptide of Suo Malu peptide is dissolved in volume fraction is to obtain thick peptide aqueous solution in 50% acetonitrile solution；
(2) 0.22 micron of organic membrane filter of the gained thick peptide solution of Suo Malu peptide, filtered solution are spare.
(3) filtered solution is taken described in taking, stationary phase is mixed into certain mass ratio with reverse phase filler and ion-exchange packing；
Detection wavelength is that 215nm carries out HPLC linear elution, wherein mobile phase A: 100mM ammonium hydrogencarbonate: the volume ratio of acetonitrile is 95:5,
Mobile phase B: 100mM ammonium hydrogencarbonate: the volume ratio of acetonitrile is 50:50；Gradient is that Initial Gradient 5%B retains 5min, then
40min to 80% collects the fraction containing Suo Malu peptide；
(4) with Rotary Evaporators revolving bath temperature at 30 DEG C to 35 DEG C, vacuum degree goes to part in -0.09Mbar with backspin
Acetonitrile obtains Suo Malu peptide aqueous solution；
(5) gained Suo Malu peptide aqueous solution -50 DEG C of pre-freezes of process, then carry out frozen dried and obtain the rope of 95% or more purity
Ma Shandong peptide sample.
The sample dissolution thick peptide of Suo Malu peptide is dissolved in acetonitrile solution, the volume ratio of acetonitrile and water in acetonitrile solution
It is 1:9 between 4:6.
Filtering filter membrane used is organic film, and the aperture of film is again between 0.1-0.5 microns.
Stationary phase used is the condiment that reverse phase is mixed in a certain proportion with ion-exchange packing, wherein reverse phase filler and ion
Packing quality ratio is exchanged between 10:1-10:5.
Reverse phase filler is common one of silica gel bonded C18, C8 filler and inverted polymer filler, and partial size is in 5-
Between 50 microns.
Organic phase packet is one of methanol, acetonitrile, ethyl alcohol, tetrahydrofuran, and wherein salt mutually includes acetate, carbonate etc.
The salt of effumability.
The elution volume of organic phase and salt phase ratio is in 95:5 between 50:50 in the gradient of linear elution.
Filtrate is taken, (10:1 is extremely with reverse phase filler (C18, C8, phenyl, C4) and ion-exchange packing with certain mass ratio
1:3) it is mixed into stationary phase；Detection wavelength is that 215nm carries out HPLC linear elution, wherein mobile phase A: 100mM ammonium hydrogencarbonate: second
The volume ratio of nitrile is 95:5, and Mobile phase B: 100mM ammonium hydrogencarbonate: the volume ratio of acetonitrile is 50:50, and gradient is Initial Gradient
5%B retains 5min, then 40min to 80%, collects the fraction containing Suo Malu peptide.
Bath temperature is rotated at 30 DEG C to 35 DEG C, vacuum degree removes partial acetonitrile in -0.09Mbar with backspin, obtains Suo Malu
Peptide aqueous solution.
Gained Suo Malu peptide aqueous solution -50 DEG C of pre-freezes of process, then carry out frozen dried and obtain the Suo Ma of 90% or more purity
Shandong peptide sample.
Compared with prior art, the positive effect of the present invention is:
(1) the application is purified by a step, so that it may which the Suo Malu peptide of purity 30% is increased to 90% or more, Er Qiechun
The change period is short, increases the production batch of purifying significantly.
(2) the application reverse phase used at present add ion-exchange packing to be mixed in a certain proportion for fixed phase stuffing performance it is steady
Fixed, carrying capacity is high, and the rate of recovery is high, and service life is permanent, regeneration easy to clean.
Detailed description of the invention
Fig. 1 is Suo Malu peptide purifying crude figure；
Fig. 2 is sample analysis figure after purification.
Specific embodiment
The specific embodiment of a kind of Suo Malu peptide purification filler special of the present invention presented below and its purification process.
Embodiment 1
A kind of Suo Malu peptide purification filler special and its purification process, its step are as follows:
(1) the thick peptide of Suo Malu peptide that 15mg purity is 30% or so is dissolved in 50% acetonitrile solution of 1.5ml, ultrasound
Dissolution, is made into concentration 10mg/ml sample solution, the organic membrane filter of the thick peptide solution 0.22um of gained Suo Malu peptide, filtered solution
It is spare；
(2) chromatographic condition: chromatographic column: reverse phase and ion-exchange packing are mixed in a certain proportion as fixed phase stuffing；Chromatography
The diameter and length of column: 4.6*250mm.Wherein mobile phase A: 100mM ammonium hydrogencarbonate: acetonitrile 95:5, Mobile phase B: 100mM carbon
Sour hydrogen ammonia: acetonitrile 50:50.Flow velocity is 1ml/min, Detection wavelength 215nm.HPLC linear elution is carried out, wherein elution ladder
Degree is that Initial Gradient 5%B retains 5min, and then 40min to 80%, collects the fraction containing Suo Malu peptide.Use Rotary Evaporators
Bath temperature is rotated at 30 DEG C to 35 DEG C, vacuum degree removes partial acetonitrile in -0.09Mbar with backspin, and it is water-soluble to obtain Suo Malu peptide
Liquid.Gained Suo Malu peptide aqueous solution -50 DEG C of pre-freezes of process, then carry out frozen dried and obtain the Suo Malu peptide sample of 90% or more purity
Product.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
Member, without departing from the inventive concept of the premise, can also make several improvements and modifications, these improvements and modifications also should be regarded as
In protection scope of the present invention.
Claims (9)
1. a kind of purification process of Suo Malu peptide purification filler special, which is characterized in that the specific steps are that:
Step 1: the thick peptide of Suo Malu peptide taken, which is dissolved in acetonitrile solution, obtains thick peptide solution, filtering；
Step 2: taking the thick peptide solution, stationary phase is mixed into reverse phase filler and ion-exchange packing, after filtrate loading, to have
Mobile phase that machine phase and salt mix while HPLC linear gradient elution is carried out, collects the fraction containing Suo Malu peptide；
Step 3: taking the third fraction through vacuum rotary steam concentration, be freeze-dried to get high-purity Suo Malu peptide.
2. a kind of purification process of Suo Malu peptide purification filler special as described in claim 1, which is characterized in that
(1) the thick peptide of Suo Malu peptide is dissolved in volume fraction is to obtain thick peptide aqueous solution in 50% acetonitrile solution；
(2) 0.22 micron of organic membrane filter of the gained thick peptide solution of Suo Malu peptide, filtered solution are spare；
(3) filtered solution is taken described in taking, stationary phase is mixed into reverse phase filler and ion-exchange packing；Detection wavelength be 215nm into
Row HPLC linear elution, wherein mobile phase A: 100mM ammonium hydrogencarbonate: the volume ratio of acetonitrile is 95:5, Mobile phase B: 100mM carbonic acid
Hydrogen ammonia: the volume ratio of acetonitrile is 50:50；Gradient is that Initial Gradient 5%B retains 5min, and then 40min to 80%, is collected
Fraction containing Suo Malu peptide；
(4) with Rotary Evaporators revolving bath temperature at 30 DEG C to 35 DEG C, vacuum degree goes part second in -0.09Mbar with backspin
Nitrile obtains Suo Malu peptide aqueous solution；
(5) gained Suo Malu peptide aqueous solution -50 DEG C of pre-freezes of process, then carry out frozen dried and obtain the Suo Malu of 95% or more purity
Peptide sample.
3. a kind of purification process of Suo Malu peptide purification filler special as described in claim 1, which is characterized in that the sample
The dissolution thick peptide of Suo Malu peptide is dissolved in acetonitrile solution, and the volume ratio of acetonitrile and water is 1:9 between 4:6 in acetonitrile solution.
4. a kind of purification process of Suo Malu peptide purification filler special as described in claim 1, which is characterized in that used in filtering
Filter membrane is organic film, and the aperture of film is again between 0.1-0.5 microns.
5. a kind of purification process of Suo Malu peptide purification filler special as described in claim 1, which is characterized in that fixation used
Phase is the condiment that reverse phase filler is mixed in a certain proportion with ion-exchange packing, wherein reverse phase filler and ion-exchange packing quality
Than between 10:1-10:5；
Reverse phase filler is common one of silica gel bonded C18, C8 filler and inverted polymer filler, and partial size is micro- in 5-50
Between rice；
Organic phase packet is one of methanol, acetonitrile, ethyl alcohol, tetrahydrofuran, and wherein salt mutually includes that acetate, carbonate etc. are easily waved
The salt of hair property.
6. a kind of purification process of Suo Malu peptide purification filler special as described in claim 1, which is characterized in that linear elution
Gradient in the elution volume of organic phase and salt phase ratio in 95:5 between 50:50.
7. a kind of purification process of Suo Malu peptide purification filler special as described in claim 1, which is characterized in that filtrate is taken,
Stationary phase is mixed into certain mass ratio 10:1 to 1:3 with reverse phase filler and ion-exchange packing；Detection wavelength is 215nm progress
HPLC linear elution, wherein mobile phase A: 100mM ammonium hydrogencarbonate: the volume ratio of acetonitrile is 95:5, Mobile phase B: 100mM bicarbonate
Ammonia: the volume ratio of acetonitrile is 50:50, and gradient is that Initial Gradient 5%B retains 5min, then 40min to 80%, and collection contains
There is the fraction of Suo Malu peptide.
8. a kind of purification process of Suo Malu peptide purification filler special as described in claim 1, which is characterized in that revolving water-bath
For temperature at 30 DEG C to 35 DEG C, vacuum degree removes partial acetonitrile in -0.09Mbar with backspin, obtains Suo Malu peptide aqueous solution.
9. a kind of purification process of Suo Malu peptide purification filler special as described in claim 1, which is characterized in that gained rope Ma
Shandong peptide aqueous solution -50 DEG C of pre-freezes of process, then carry out frozen dried and obtain the Suo Malu peptide sample of 90% or more purity.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020190757A1 (en) * | 2019-03-15 | 2020-09-24 | Novetide Ltd. | Improved processes for the preparation of semaglutide |
| CN111848777A (en) * | 2020-08-17 | 2020-10-30 | 深圳瑞德林生物技术有限公司 | Method for purifying somaglutide |
| CN112175068A (en) * | 2020-09-28 | 2021-01-05 | 深圳深创生物药业有限公司 | Method for purifying semaglutide |
| CN112279907A (en) * | 2019-07-27 | 2021-01-29 | 深圳市健元医药科技有限公司 | Purification method of somaglutide |
| WO2021129308A1 (en) * | 2019-12-27 | 2021-07-01 | 翰宇药业（武汉）有限公司 | Method for purifying glp-1 analog |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2020190757A1 (en) * | 2019-03-15 | 2020-09-24 | Novetide Ltd. | Improved processes for the preparation of semaglutide |
| CN112279907A (en) * | 2019-07-27 | 2021-01-29 | 深圳市健元医药科技有限公司 | Purification method of somaglutide |
| 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 |
| WO2021129308A1 (en) * | 2019-12-27 | 2021-07-01 | 翰宇药业（武汉）有限公司 | Method for purifying glp-1 analog |
| CN111848777A (en) * | 2020-08-17 | 2020-10-30 | 深圳瑞德林生物技术有限公司 | Method for purifying somaglutide |
| CN112175068A (en) * | 2020-09-28 | 2021-01-05 | 深圳深创生物药业有限公司 | Method for purifying semaglutide |
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