CN106632608A - Purifying method for arigireline - Google Patents
Purifying method for arigireline Download PDFInfo
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- CN106632608A CN106632608A CN201710005787.4A CN201710005787A CN106632608A CN 106632608 A CN106632608 A CN 106632608A CN 201710005787 A CN201710005787 A CN 201710005787A CN 106632608 A CN106632608 A CN 106632608A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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Abstract
The invention provides a purifying method for arigireline. The purifying method comprises the following steps: (1) dissolving: dissolving a crude arigireline product to be purified in a solution of acetic acid-sodium acetate and carrying out filtering so as to obtain a filtrate; (2) rough purification: loading the filtrate obtained in the step (1) into a strong cation exchange column, carrying out gradient eluation by using a rough purification mobile phase and collecting eluate; and (3) desalination: loading the eluate obtained in the step (2) into a reversed-phase polymer column, carrying out gradient eluation by using a desalination mobile phase, collecting an arigireline solution and carrying out pressure-reduced concentration and freeze drying so as to obtain arigireline.
Description
Technical field
The invention belongs to polypeptide purification techniques field, in particular it relates to a kind of purification process of Argireline.
Background technology
Argireline is also known as hexa-atomic victory peptide, the repertoire with creotoxin, and nontoxicity.Argireline is a kind of containing six
The active peptides of amino acid, amino acid sequence is Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2, and it can suppress nerve conduction,
Muscle excess shrinkage is avoided, so as to prevent microgroove from being formed.As skin wrinkle composition, it is widely used in various senior beauty and make-ups
In product.Compact stoste in the MAGIC CARE magics nursing flat wrinkle of Argireline of research and development in 2010 in Switzerland legend group skin care laboratory
It is the tip representative of Argireline cosmetics.In addition, the pertinent literature of domestic and international rare Argireline isolation and purification method, does not also have
Document specifically studies the related large-scale isolation and purification method of Argireline.
The content of the invention
The technical problem to be solved is the shortcoming for overcoming existing Argireline purification process to exist, there is provided a kind of
Low cost, purity are high, are adapted to the Argireline purification process of industrialization.
The purification process of the Argireline that the present invention is provided includes：
(1) dissolve：By Argireline dissolving crude product to be purified in Acetic acid-sodium acetate solution, filtrate is filtrated to get；
(2) it is thick pure：The filtrate loading that step (1) is obtained carries out gradient to strong cat ion exchange column with thick pure mobile phase
Wash-out, collects eluent；
(3) desalination：The eluent loading that step (2) is collected carries out gradient to inverted polymer post with desalination mobile phase
Wash-out, collects Argireline solution, and reduced pressure concentration obtains Argireline with freeze-drying.
Aforesaid purification process, in step (1), Argireline crude product and the ratio of Acetic acid-sodium acetate solution to be purified is
(0.5g-100g):(20ml-1000ml), it is preferable that Argireline crude product and the ratio of Acetic acid-sodium acetate solution to be purified be
(0.5g-10g):(20ml-100ml)。
Aforesaid purification process, it is 3.9-4.1 for 0.01-0.1mol/L, pH value that the Acetic acid-sodium acetate solution is concentration
Acetic acid-sodium acetate solution.
Aforesaid purification process, the thick pure mobile phase is by initial buffer liquid mobile phase and elution buffer mobile phase group
Into, wherein, the initial buffer liquid mobile phase is Acetic acid-sodium acetate solution, and the elution buffer mobile phase is to add chlorination
The initial buffer liquid mobile phase of sodium；Preferably, the initial buffer liquid mobile phase is that concentration is for 0.01-0.1mol/L, pH value
The Acetic acid-sodium acetate solution of 3.9-4.1, the elution buffer mobile phase is to add the initial of 0.5-1.5mol/L sodium chloride to delay
Rush liquid mobile phase.
Aforesaid purification process, the desalination mobile phase is made up of mobile phase A and Mobile phase B, wherein, the mobile phase A
It is ultra-pure water, the Mobile phase B is Chromatographic Pure Methanol.
Aforesaid purification process, in step (2), the thick pure eluent gradient is initial buffer liquid mobile phase:Wash-out is slow
Liquid mobile phase is rushed by 100:0 arrives (80-60):(20-40).
Aforesaid purification process, in step (3), the desalination eluent gradient is mobile phase A:Mobile phase B is by (100-
95):(0-5) (90-80) is arrived:(10-20).
Aforesaid purification process, the thick pure mobile phase is by initial buffer liquid mobile phase and elution buffer mobile phase group
Into, wherein, the initial buffer liquid mobile phase is Acetic acid-sodium acetate solution, and the elution buffer mobile phase is high concentration, height
The Acetic acid-sodium acetate buffer solution of pH value；Preferably, the initial buffer liquid mobile phase is that concentration is 0.01-0.1mol/L, pH value
For the Acetic acid-sodium acetate solution of 3.9-4.1, by A phases and B phase compositions, A phases are that concentration is to the elution buffer mobile phase
0.01-0.1mol/L, pH value are the Acetic acid-sodium acetate solution of 5.5-6.2, and B phases are that concentration is for 0.5-1.5mol/L, pH value
5.5-6.2 Acetic acid-sodium acetate solution.
Aforesaid purification process, the desalination mobile phase is made up of mobile phase A and Mobile phase B, wherein, the mobile phase A
It is ultra-pure water, the Mobile phase B is Chromatographic Pure Methanol.
Aforesaid purification process, in step (2), the thick pure eluent gradient is initial buffer liquid mobile phase:Wash-out is slow
Liquid mobile phase is rushed by 100:0 arrives (80-60):(20-40).
Aforesaid purification process, in step (3), the desalination eluent gradient is mobile phase A:Mobile phase B is by (100-
95):(0-5) (90-80) is arrived:(5-10).
Using technical scheme, at least have the advantages that：
1. the inventive method is used in combination strong cat ion exchange column with inverted polymer post, first using strong cation exchange
Post carries out thick pure to Argireline, eliminates most of impurity, and reusing inverted polymer post carries out desalination, is further purified
Argireline, drastically increase purification efficiency.
2. the inventive method saves the cost of mobile phase in whole process of purification, and purifying process is more environmentally friendly.
3. two kinds of pillars of the inventive method are used alternatingly, and effectively compensate for single pillar and are difficult to be kept completely separate in thick peptide not
The impurity of same structure, different chemical property, the Argireline purity for obtaining high (more than 95%), and high income.
Description of the drawings
Fig. 1 is the mass spectrogram of the Argireline obtained according to the method purifying of the first specific embodiment of the invention.
Fig. 2 is the high performance liquid chromatography of the Argireline obtained according to the method purifying of the first specific embodiment of the invention
Figure.
Fig. 3 is the mass spectrogram of the Argireline obtained according to the method for second specific embodiment of the invention purifying.
Fig. 4 is the high performance liquid chromatography of the Argireline obtained according to the method for second specific embodiment of the invention purifying
Figure.
Specific embodiment
To be fully understood by purpose, feature and effect of the present invention, by following specific embodiments, the present invention is done in detail
Describe in detail bright, but the present invention is not restricted to this.
At present, for Argireline purification process, commonly there is a problem of that with high costs but effect is undesirable, ask for this
Topic, the invention provides a kind of method that utilization Ion-exchange high-performance liquid chromatography isolates and purifies Argireline, the method is wrapped successively
Include following steps：
(1) dissolve：By Argireline dissolving crude product to be purified in Acetic acid-sodium acetate solution, filtrate is filtrated to get；
(2) it is thick pure：The filtrate loading that step (1) is obtained carries out gradient to strong cat ion exchange column with thick pure mobile phase
Wash-out, collects eluent；
(3) desalination：The eluent loading that step (2) is collected carries out gradient to inverted polymer post with desalination mobile phase
Wash-out, collects Argireline solution, and reduced pressure concentration obtains Argireline with freeze-drying.
In step (1), Argireline crude product to be purified is obtained using solid-phase synthesis, by Argireline to be purified
Dissolving crude product is when Acetic acid-sodium acetate solution, it is preferable that according to Argireline crude product to be purified and Acetic acid-sodium acetate solution
Ratio is (0.5g-100g):(20ml-1000ml) dissolved, it is highly preferred that according to Argireline crude product and vinegar to be purified
The ratio of acid-SAS is (0.5g-10g):(20ml-100ml) dissolved.By Argireline dissolving crude product to be purified
After Acetic acid-sodium acetate solution, it is preferred to use 1mol/L NaOH adjust pH value to 3.9-4.1, carry out it is ultrasonically treated, subsequently
Filtered with filter membrane (such as 0.45 μm of filter membrane), and collect filtrate and do not used.
In step (2), chromatographic column adopts strong cat ion exchange column, so-called strong cat ion exchange column to refer to highly purified
Inertia Silica Surface is bonded a kind of new sulfonic acids base cation exchange ligand, and this unique bonding structure is a kind of
Stable polymerization capsule structure, it is higher than common polymer ions exchange column post effect, and with more preferable mechanical strength and weight
Existing property.In the present invention, the strong cat ion exchange column for adopting is filler for the strong cat ion exchange column of agarose microbeads, wherein fine jade
Lipolysaccharide microballoon can be SP high flow rate agarose microbeads (SP Bio-sepFF), and its particle diameter is 50-160 μm, can be by conventional city
Available from the SP high flow rates agarose microbeads of such as Bio-sep Bio-technique Stock Co., Ltd. Xi'an Jiaotong University's production just can be used for this
In invention.
In step (3), chromatographic column adopts inverted polymer post, so-called inverted polymer post to refer to the polarity ratio of fixing phase
The polarity of mobility is weak one to birds of the same feather flock together compound post.In the present invention, the inverted polymer post for adopting is that filler is styrene diethyl
The inverted polymer post of alkene benzene-type filler, such as filler are SBC MCI GEI F type reverse-phase chromatography fillers, particle diameter is 30-50 μm,
Can be by conventional city available from the SBC MCI GEI F type reverse-phase chromatography fillers of the biological Co., Ltd's production of such as Chengdu section spectrum
Can be used in the present invention.
Thick pure mobile phase used in step (2) is by initial buffer liquid mobile phase and elution buffer flowing phase composition, step
Suddenly the desalination mobile phase used in (3) is made up of mobile phase A and Mobile phase B.
The present invention the first specific embodiment in, the initial buffer liquid mobile phase used in step (2) be acetic acid-
SAS, elution buffer mobile phase is the initial buffer liquid mobile phase for adding sodium chloride；Preferably, initial buffer liquid stream
Dynamic phase is that concentration is 0.01-0.1mol/L, the Acetic acid-sodium acetate solution that pH value is 3.9-4.1, and elution buffer mobile phase is to add
Enter the initial buffer liquid mobile phase of 0.5-1.5mol/L sodium chloride.Mobile phase A used in step (3) is ultra-pure water, Mobile phase B
It is Chromatographic Pure Methanol.
In step (2), by the filtrate loading of step (1) collection to before strong cat ion exchange column, first by strong cation
It is 3.9-4.1 that the flowing of exchange column initial buffer liquid balances each other to detector efflux pH value, and electrical conductivity is invariable.By filtrate
Loading to strong cat ion exchange column, according to initial buffer liquid mobile phase:Elution buffer mobile phase is by 100:0 arrives (80-
60):(20-40) gradient carries out gradient elution.For example, elution process can be divided into the different stages, for example, 0-60 minutes
For the first stage, 60-90 minutes be second stage, 90-150 minutes be the phase III, or 0-80 minutes be the first stage,
80-120 minutes be second stage, 120-240 minutes be the phase III, or 0-110 minutes are first stage, 110-170 point
Clock be second stage, 170-340 minutes be the phase III, or 0-120 minutes be the first stage, 120-180 minutes be second
Stage, 180-340 minutes are the phase III, and the gradient of elution process can be initial buffer liquid mobile phase in the first stage
100%, initial buffer liquid mobile phase in second stage:Elution buffer mobile phase is by (100-70)：(0-30) (70-65) is arrived：
(30-35), phase III initial buffer liquid mobile phase:Elution buffer mobile phase is (70-65)：(30-35) constant current.Gradient is washed
It is de-, collect eluent.
In step (3), by the eluent loading of step (2) collection to before inverted polymer post, first by reversed-phase polymerization
Thing post is balanced with mobile phase A.After by eluent loading to inverted polymer post, according to mobile phase A:Mobile phase B by
(100-95):(0-5) (90-80) is arrived:(10-20) gradient carries out gradient elution.For example, elution process can be divided into difference
Stage, for example, 0-45 minutes be the first stage, 45-100 minutes be second stage, or 0-50 minutes be the first stage,
50-100 minutes are second stage, or it first stage, 80-160 minutes is second stage that 0-80 minutes are, or 0-100 point
Clock is the first stage, 100-200 minutes are second stage, and the gradient of elution process can be first stage mobile phase A：Mobile phase
B is by (100-95):(0-5) (90-80) is arrived:(10-20), second stage mobile phase A:Mobile phase B is (90-80):(10-20) it is permanent
Stream.Gradient elution, collects the peptide solution of purpose peak value, by the purpose peptide solution after desalination less than 40 DEG C of reduced pressure concentrations, concentration
Freeze to about 50mg-100mg/mL, that is, obtain the Argireline certified products pressed powder of purity more than 95%, its mass spectrogram such as Fig. 1
Shown, high-efficient liquid phase chromatogram is as shown in Figure 2.
The present invention second specific embodiment in, the initial buffer liquid mobile phase used in step (2) be acetic acid-
SAS, the elution buffer mobile phase is the Acetic acid-sodium acetate buffer solution of high concentration, high ph-values；Preferably, it is described
Initial buffer liquid mobile phase is that concentration is 0.01-0.1mol/L, the Acetic acid-sodium acetate solution that pH value is 3.9-4.1, the wash-out
By A phases and B phase compositions, A phases are that concentration is 0.01-0.1mol/L, acetic acid-acetic acid that pH value is 5.5-6.2 to buffer solution mobile phase
Sodium solution, B phases are that concentration is 0.5-1.5mol/L, the Acetic acid-sodium acetate solution that pH value is 5.5-6.2.Used in step (3)
Mobile phase A is ultra-pure water, and Mobile phase B is Chromatographic Pure Methanol.
In step (2), by the filtrate loading of step (1) collection to before strong cat ion exchange column, first by strong cation
It is 3.9-4.1 that the flowing of exchange column initial buffer liquid balances each other to detector efflux pH value, and electrical conductivity is invariable.By filtrate
Loading to strong cat ion exchange column, according to initial buffer liquid mobile phase:Elution buffer mobile phase is by 100:0 arrives (80-
60):(20-40) gradient carries out gradient elution.For example, elution process can be divided into the different stages, for example, 0-60 minutes
For the first stage, 60-90 minutes be second stage, 90-150 minutes be the phase III, or 0-80 minutes be the first stage,
80-120 minutes be second stage, 120-240 minutes be the phase III, or 0-110 minutes are first stage, 110-170 point
Clock be second stage, 170-340 minutes be the phase III, or 0-120 minutes be the first stage, 120-180 minutes be second
Stage, 180-340 minutes are the phase III, and the gradient of elution process can be initial buffer liquid mobile phase in the first stage
100%, initial buffer liquid mobile phase in second stage:Elution buffer mobile phase is by (100-70)：(0-30) (70-65) is arrived：
(30-35), phase III initial buffer liquid mobile phase:Elution buffer mobile phase is (70-65)：(30-35) constant current.Gradient is washed
It is de-, collect eluent.
In step (3), by the eluent loading of step (2) collection to before inverted polymer post, first by reversed-phase polymerization
Thing post is balanced with mobile phase A.After by eluent loading to inverted polymer post, according to mobile phase A:Mobile phase B by
(100-95):(0-5) (90-80) is arrived:(10-20) gradient carries out gradient elution.For example, elution process can be divided into difference
Stage, for example, 0-45 minutes be the first stage, 45-100 minutes be second stage, or 0-50 minutes be the first stage,
50-100 minutes are second stage, or it first stage, 80-160 minutes is second stage that 0-80 minutes are, or 0-100 point
Clock is the first stage, 100-200 minutes are second stage, and the gradient of elution process can be first stage mobile phase A:Mobile phase
B is by (100-98):(0-2) (95-90) is arrived:(5-10), second stage mobile phase A:Mobile phase B is (95-90):(5-10) it is permanent
Stream.Gradient elution, collects the peptide solution of purpose peak value, by the purpose peptide solution after desalination less than 40 DEG C of reduced pressure concentrations, concentration
Freeze to about 50mg-100mg/mL, that is, obtain the Argireline certified products pressed powder of purity more than 95%, its mass spectrogram such as Fig. 3
Shown, high-efficient liquid phase chromatogram is as shown in Figure 4.
With reference to the accompanying drawings and examples the present invention is described in more detail, but the present invention is not limited to these enforcements
Example.Unless otherwise stated, the raw material and instrument used in embodiment is commercially available, is instrument commonly used in the art
And raw material, as long as it can meet experiment needs.
Embodiment 1
1. molten sample
0.5g synthesis in solid state gained Argireline is weighed, it is molten with 20ml, pH=4,0.01mol/L Acetic acid-sodium acetate aqueous solution
Solution (concentration is about 25mg/mL), with 1mol/L NaOH pH=3.9-4.1 is adjusted, ultrasonically treated, with 0.45 μm of membrane filtration,
Collect filtrate standby.
2. thick pure
Purification condition：Chromatographic column：Filler be SP high flow rate agarose microbeads Bio-sepFF, the strong sun of 50-160 μm of particle diameter
Ion exchange column, it is 50mL that pillar loads volume.Initial buffer liquid mobile phase is：0.01mol/L pH=3.9-4.1 acetic acid-vinegar
Acid sodium aqueous solution, elution buffer mobile phase is the initial buffer liquid mobile phase for adding 1mol/L sodium chloride.Flow velocity 5mL/min.
Detection wavelength：215nm.Gradient：0 to 60 minutes initial buffer liquid mobile phases 100%, initial buffer liquid flowing in 60 to 90 minutes
Phase:Elution buffer mobile phase is by 100:0 to 70:30, it is within 90 to 150 minutes initial buffer liquid mobile phase:Elution buffer stream
Dynamic is mutually 70:30 constant currents.
Purge process：Cation exchange column is balanced each other to detector efflux pH=with the flowing of 100% initial buffer liquid
3.9-4.1, the invariable loading of electrical conductivity.Linear gradient elution, the peptide solution for collecting purpose peak value is stand-by.
3. desalination
Purification condition：Chromatographic column：Filler be SBC MCI GEI F type 30-50 μm reverse-phase chromatography filler F types 30-50 μm, post
It is 50mL that son loads volume.Mobile phase A is：Ultra-pure water, Mobile phase B is：Chromatographic Pure Methanol.Flow velocity 7mL/min.Detection wavelength:
215nm.Gradient：0 to 50 minutes mobile phase As:Mobile phase B is by 100:0 to 90:10, it is within 50 to 100 minutes mobile phase A:Mobile phase
B is 90:10 constant currents.
Purge process：By polymer column with the ultrapure water balance of 100% mobile phase A well after loading.Linear gradient elution, collects
The peptide solution of purpose peak value, by the purpose peptide solution after desalination less than 40 DEG C of reduced pressure concentrations, is concentrated into about 50mg-100mg/mL
After freeze, that is, obtain the salt acid type Argireline certified products pressed powder of purity 97.4%.
Embodiment 2
1. molten sample
1.0g synthesis in solid state gained Argireline is weighed, it is molten with 50ml, pH=4,0.01mol/L acetic acid/sodium acetate aqueous solution
Solution (concentration is about 20mg/mL), with 1mol/L NaOH pH=3.9-4.1 is adjusted, ultrasonically treated, with 0.45 μm of membrane filtration,
Collect filtrate standby.
2. thick pure
Purification condition：Chromatographic column：Filler be SP high flow rate agarose microbeads Bio-sepFF, the strong sun of 50-160 μm of particle diameter
Ion exchange column, it is 150mL that pillar loads volume.Initial buffer liquid mobile phase is：0.01mol/L pH=3.9-4.1 acetic acid-
Sodium acetate aqueous solution, elution buffer mobile phase is the initial buffer liquid mobile phase for adding 1mol/L sodium chloride.Flow velocity 10mL/
min.Detection wavelength：215nm.Gradient：0 to 80 minutes initial buffer liquid mobile phases 100%, 80 to 120 minutes initial buffer liquid
Mobile phase:Elution buffer mobile phase is by 100:0 to 70:30, it is within 120 to 240 minutes initial buffer liquid mobile phase:Elution buffer
Liquid mobile phase is 70:30 constant currents.
Purge process：Cation exchange column is balanced each other to detector efflux pH=with the flowing of 100% initial buffer liquid
3.9-4.1, the invariable loading of electrical conductivity.Linear gradient elution, the peptide solution for collecting purpose peak value is stand-by.
3. desalination
Purification condition：Chromatographic column：Filler be SBC MCI GEI F type 30-50 μm reverse-phase chromatography filler F types 30-50 μm, post
It is 150mL that son loads volume.Mobile phase A is：Ultra-pure water, Mobile phase B is：Chromatographic Pure Methanol.Flow velocity 20mL/min.Detection ripple
It is long：215nm.Gradient：0 to 80 minutes mobile phase As:Mobile phase B is by 100:0 to 90:10, it is within 80 to 160 minutes mobile phase A:Stream
Dynamic phase B is 90:10 constant currents.
Purge process：By polymer column with the ultrapure water balance of 100% mobile phase A well after loading.Linear gradient elution, collects
The peptide solution of purpose peak value, by the purpose peptide solution after desalination less than 40 DEG C of reduced pressure concentrations, is concentrated into about 50mg-100mg/mL
After freeze, that is, obtain the salt acid type Argireline certified products pressed powder of purity 97%.
Embodiment 3
1. molten sample
30.0g synthesis in solid state gained Argireline is weighed, with 300ml, pH=4,0.01mol/L Acetic acid-sodium acetate aqueous solution
Dissolving (concentration is about 100mg/mL), with 1mol/L NaOH pH=3.9-4.1 is adjusted, ultrasonically treated, with 0.45 μm of filter membrane mistake
Filter, collects filtrate standby.
2. thick pure
Purification condition：Chromatographic column：Filler be SP high flow rate agarose microbeads Bio-sepFF, the strong sun of 50-160 μm of particle diameter
Ion exchange column, it is 2500mL that pillar loads volume.Initial buffer liquid mobile phase is：0.01M pH=3.9-4.1 acetic acid-acetic acid
Sodium water solution, elution buffer mobile phase is the initial buffer liquid mobile phase for adding 1mol/L sodium chloride.Flow velocity 25mL/min.Inspection
Survey wavelength：215nm.Gradient：0 to 110 minutes initial buffer liquid mobile phases 100%, initial buffer liquid flowing in 110 to 170 minutes
Phase：Elution buffer mobile phase is by 75:25 to 70:30, it is within 170 to 340 minutes initial buffer liquid mobile phase：Elution buffer stream
Dynamic is mutually 70:30 constant currents.
Purge process：Cation exchange column is balanced each other to detector efflux pH=with the flowing of 100% initial buffer liquid
3.9-4.1, the invariable loading of electrical conductivity.Linear gradient elution, the peptide solution for collecting purpose peak value is stand-by.
3. desalination
Purification condition：Chromatographic column：Filler be SBC MCI GEI F type 30-50 μm reverse-phase chromatography filler F types 30-50 μm, post
It is 300mL that son loads volume.Mobile phase A is：Ultra-pure water, Mobile phase B is：Chromatographic Pure Methanol.Flow velocity 30mL/min.Detection ripple
It is long:215nm.Gradient：0 to 45 minutes mobile phase As：Mobile phase B is by 100:0 to 90:10, it is within 45 to 100 minutes mobile phase A：Stream
Dynamic phase B is 90:10 constant currents.
Purge process：By polymer column with the ultrapure water balance of 100% mobile phase A well after loading.Linear gradient elution, collects
The peptide solution of purpose peak value, by the purpose peptide solution after desalination less than 40 DEG C of reduced pressure concentrations, is concentrated into about 50mg-100mg/mL
After freeze, that is, obtain the salt acid type Argireline certified products pressed powder of purity 96.2%.
Embodiment 4
1. molten sample
60.0g synthesis in solid state gained Argireline is weighed, with 600ml, pH=4,0.01mol/L Acetic acid-sodium acetate aqueous solution
Dissolving (concentration is about 100mg/mL), with 1mol/L NaOH pH=3.9-4.1 is adjusted, ultrasonically treated, with 0.45 μm of filter membrane mistake
Filter, collects filtrate standby.
2. thick pure
Purification condition：Chromatographic column：Filler be SP high flow rate agarose microbeads Bio-sepFF, the strong sun of 50-160 μm of particle diameter
Ion exchange column, it is 2500mL that pillar loads volume.Initial buffer liquid mobile phase is：0.01mol/L pH=3.9-4.1 acetic acid-
Sodium acetate aqueous solution, elution buffer mobile phase is the initial buffer liquid mobile phase for adding 1mol/L sodium chloride.Flow velocity 25mL/
min.Detection wavelength:215nm.Gradient：0 to 110 minutes initial buffer liquid mobile phases 100%, 110 to 170 minutes initial buffers
Liquid mobile phase：Elution buffer mobile phase is by 70:30 to 65:35, it is within 170 to 340 minutes initial buffer liquid mobile phase：Wash-out is slow
Liquid mobile phase is rushed for 65:35 constant currents.
Purge process：Cation exchange column is balanced each other to detector efflux pH=with the flowing of 100% initial buffer liquid
3.9-4.1, the invariable loading of electrical conductivity.Linear gradient elution, the peptide solution for collecting purpose peak value is stand-by.
3. desalination
Purification condition：Chromatographic column：Filler be SBC MCI GEI F type 30-50 μm reverse-phase chromatography filler F types 30-50 μm, post
It is 3000mL that son loads volume.Mobile phase A is：Ultra-pure water, Mobile phase B is：Chromatographic Pure Methanol.Flow velocity 50mL/min.Detection ripple
It is long：215nm.Gradient：0 to 100 minutes mobile phase As：Mobile phase B is by 100:0 to 90:15, it is within 100 to 200 minutes mobile phase A:
Mobile phase B is 90:15 constant currents.
Purge process：By polymer column with the ultrapure water balance of 100% mobile phase A well after loading.Linear gradient elution, collects
The peptide solution of purpose peak value, by the purpose peptide solution after desalination less than 40 DEG C of reduced pressure concentrations, is concentrated into about 50mg-100mg/mL
After freeze, that is, obtain the salt acid type Argireline certified products pressed powder of purity 96%.
Embodiment 5
1. molten sample
100.0g synthesis in solid state gained Argireline is weighed, it is water-soluble with 1000ml, pH=4,0.01mol/L Acetic acid-sodium acetate
Liquid dissolves (concentration is about 100mg/mL), and with 1mol/L NaOH pH=3.9-4.1 is adjusted, ultrasonically treated, with 0.45 μm of filter membrane
Filter, collect filtrate standby.
2. thick pure
Purification condition：Chromatographic column：Filler be SP high flow rate agarose microbeads Bio-sepFF, the strong sun of 50-160 μm of particle diameter
Ion exchange column, it is 2500mL that pillar loads volume.Initial buffer liquid mobile phase is：0.01mol/L pH=3.9-4.1 acetic acid-
Sodium acetate aqueous solution, elution buffer mobile phase is the initial buffer liquid mobile phase for adding 1mol/L sodium chloride.Flow velocity 25mL/
min.Detection wavelength：215nm.Gradient：0 to 120 minutes initial buffer liquid mobile phases 100%, 120 to 180 minutes initial buffers
Liquid mobile phase：Elution buffer mobile phase is by 70:30 to 65:35, it is within 180 to 340 minutes initial buffer liquid mobile phase：Wash-out is slow
Liquid mobile phase is rushed for 65:35 constant currents.
Purge process：Cation exchange column is balanced each other to detector efflux pH=with the flowing of 100% initial buffer liquid
3.9-4.1, the invariable loading of electrical conductivity.Linear gradient elution, the peptide solution for collecting purpose peak value is stand-by.
3. desalination
Purification condition：Chromatographic column：Filler be SBC MCI GEI F type 30-50 μm reverse-phase chromatography filler F types 30-50 μm, post
It is 3000mL that son loads volume.Mobile phase A is：Ultra-pure water, Mobile phase B is：Chromatographic Pure Methanol.Flow velocity 50mL/min.Detection ripple
It is long：215nm.Gradient：0 to 100 minutes mobile phase As：Mobile phase B is by 100:0 to 90:15, it is within 100 to 200 minutes mobile phase A：
Mobile phase B is 90:15 constant currents.
Purge process:By polymer column with the ultrapure water balance of 100% mobile phase A well after loading.Linear gradient elution, collects
The peptide solution of purpose peak value, by the purpose peptide solution after desalination less than 40 DEG C of reduced pressure concentrations, is concentrated into about 50mg-100mg/mL
After freeze, that is, obtain the salt acid type Argireline certified products pressed powder of purity 96%.
Embodiment 6
1. molten sample
0.5g synthesis in solid state gained Argireline is weighed, it is molten with 20ml, pH=4,0.01mol/L Acetic acid-sodium acetate aqueous solution
Solution (concentration is about 25mg/mL), with 1mol/L NaOH pH=3.9-4.1 is adjusted, ultrasonically treated, with 0.45 μm of membrane filtration,
Collect filtrate standby.
2. thick pure
Purification condition：Chromatographic column：Filler be SP high flow rate agarose microbeads Bio-sepFF, the strong sun of 50-160 μm of particle diameter
Ion exchange column, it is 50mL that pillar loads volume.Initial buffer liquid mobile phase is：0.01mol/L pH=3.9-4.1 acetic acid-vinegar
Acid sodium aqueous solution, elution buffer mobile phase is A phases：The Acetic acid-sodium acetate that concentration is 0.01mol/L, pH value is 5.5-6.2 is molten
Liquid, B phases：The Acetic acid-sodium acetate solution that concentration is 1mol/L, pH value is 5.5-6.2, flow velocity 5mL/min.Detection wavelength：
215nm.Gradient：0 to 60 minutes initial buffer liquid mobile phases 100%, 60 to 90 minutes initial buffer liquid mobile phases:Elution buffer
Liquid mobile phase is by 100:0 to 70:30, it is within 90 to 150 minutes initial buffer liquid mobile phase:Elution buffer mobile phase is 70:30
Constant current.
Purge process：Cation exchange column is balanced each other to detector efflux pH=with the flowing of 100% initial buffer liquid
3.9-4.1, the invariable loading of electrical conductivity.Linear gradient elution, the peptide solution for collecting purpose peak value is stand-by.
3. desalination
Purification condition：Chromatographic column：Filler be SBC MCI GEI F type 30-50 μm reverse-phase chromatography filler F types 30-50 μm, post
It is 50mL that son loads volume.Mobile phase A is：Ultra-pure water, Mobile phase B is：Chromatographic Pure Methanol.Flow velocity 7mL/min.Detection wavelength:
215nm.Gradient：0 to 50 minutes mobile phase As:Mobile phase B is by 100:0 to 90:10, it is within 50 to 100 minutes mobile phase A:Mobile phase
B is 90:10 constant currents.
Purge process：By polymer column with the ultrapure water balance of 100% mobile phase A well after loading.Linear gradient elution, collects
The peptide solution of purpose peak value, by the purpose peptide solution after desalination less than 40 DEG C of reduced pressure concentrations, is concentrated into about 50mg-100mg/mL
After freeze, that is, obtain the acetic acid type Argireline certified products pressed powder of purity 97%.
Embodiment 7
1. molten sample
1.0g synthesis in solid state gained Argireline is weighed, it is molten with 50ml, pH=4,0.01mol/L acetic acid/sodium acetate aqueous solution
Solution (concentration is about 20mg/mL), with 1mol/L NaOH pH=3.9-4.1 is adjusted, ultrasonically treated, with 0.45 μm of membrane filtration,
Collect filtrate standby.
2. thick pure
Purification condition：Chromatographic column：Filler be SP high flow rate agarose microbeads Bio-sepFF, the strong sun of 50-160 μm of particle diameter
Ion exchange column, it is 150mL that pillar loads volume.Initial buffer liquid mobile phase is：0.01mol/L pH=3.9-4.1 acetic acid-
Sodium acetate aqueous solution, elution flow is mutually A phases：The Acetic acid-sodium acetate solution that concentration is 0.01mol/L, pH value is 5.5-6.2, B
Phase：The Acetic acid-sodium acetate solution that concentration is 1.0mol/L, pH value is 5.5-6.2, flow velocity 10mL/min.Detection wavelength：215nm.
Gradient：0 to 80 minutes initial buffer liquid mobile phases 100%, 80 to 120 minutes initial buffer liquid mobile phases:Elution buffer stream
Move by 100:0 to 70:30, it is within 120 to 240 minutes initial buffer liquid mobile phase:Elution buffer mobile phase is 70:30 is permanent
Stream.
Purge process：Cation exchange column is balanced each other to detector efflux pH=with the flowing of 100% initial buffer liquid
3.9-4.1, the invariable loading of electrical conductivity.Linear gradient elution, the peptide solution for collecting purpose peak value is stand-by.
3. desalination
Purification condition：Chromatographic column：Filler be SBC MCI GEI F type 30-50 μm reverse-phase chromatography filler F types 30-50 μm, post
It is 150mL that son loads volume.Mobile phase A is：Ultra-pure water, Mobile phase B is：Chromatographic Pure Methanol.Flow velocity 20mL/min.Detection ripple
It is long：215nm.Gradient：0 to 80 minutes mobile phase As:Mobile phase B is by 100:0 to 90:10, it is within 80 to 160 minutes mobile phase A:Stream
Dynamic phase B is 90:10 constant currents.
Purge process：By polymer column with the ultrapure water balance of 100% mobile phase A well after loading.Linear gradient elution, collects
The peptide solution of purpose peak value, by the purpose peptide solution after desalination less than 40 DEG C of reduced pressure concentrations, is concentrated into about 50mg-100mg/mL
After freeze, that is, obtain the acetic acid type Argireline certified products pressed powder of purity 96.5%.
Embodiment 8
1. molten sample
30.0g synthesis in solid state gained Argireline is weighed, with 300ml, pH=4,0.01mol/L Acetic acid-sodium acetate aqueous solution
Dissolving (concentration is about 100mg/mL), with 1mol/L NaOH pH=3.9-4.1 is adjusted, ultrasonically treated, with 0.45 μm of filter membrane mistake
Filter, collects filtrate standby.
2. thick pure
Purification condition：Chromatographic column：Filler be SP high flow rate agarose microbeads Bio-sepFF, the strong sun of 50-160 μm of particle diameter
Ion exchange column, it is 2500mL that pillar loads volume.Initial buffer liquid mobile phase is：0.01M pH=3.9-4.1 acetic acid-acetic acid
Sodium water solution, elution flow is mutually A phases：The Acetic acid-sodium acetate solution that concentration is 0.01mol/L, pH value is 5.5-6.2, B phases：
The Acetic acid-sodium acetate solution that concentration is 1.0mol/L, pH value is 5.5-6.2, flow velocity 25mL/min.Detection wavelength：215nm.Ladder
Degree：0 to 110 minutes initial buffer liquid mobile phases 100%, 110 to 170 minutes initial buffer liquid mobile phases：Elution buffer stream
Move by 75:25 to 70:It is within 30,170 to 340 minutes initial buffer liquid mobile phase：Elution buffer mobile phase is 70:30 is permanent
Stream.
Purge process：Cation exchange column is balanced each other to detector efflux pH=with the flowing of 100% initial buffer liquid
3.9-4.1, the invariable loading of electrical conductivity.Linear gradient elution, the peptide solution for collecting purpose peak value is stand-by.
3. desalination
Purification condition：Chromatographic column：Filler be SBC MCI GEI F type 30-50 μm reverse-phase chromatography filler F types 30-50 μm, post
It is 300mL that son loads volume.Mobile phase A is：Ultra-pure water, Mobile phase B is：Chromatographic Pure Methanol.Flow velocity 30mL/min.Detection ripple
It is long:215nm.Gradient：0 to 45 minutes mobile phase As：Mobile phase B is by 100:0 to 90:10, it is within 45 to 100 minutes mobile phase A：Stream
Dynamic phase B is 90:10 constant currents.
Purge process：By polymer column with the ultrapure water balance of 100% mobile phase A well after loading.Linear gradient elution, collects
The peptide solution of purpose peak value, by the purpose peptide solution after desalination less than 40 DEG C of reduced pressure concentrations, is concentrated into about 50mg-100mg/mL
After freeze, that is, obtain the acetic acid type Argireline certified products pressed powder of purity 96.3%.
Embodiment 9
1. molten sample
60.0g synthesis in solid state gained Argireline is weighed, with 600ml, pH=4,0.01mol/L Acetic acid-sodium acetate aqueous solution
Dissolving (concentration is about 100mg/mL), with 1mol/L NaOH pH=3.9-4.1 is adjusted, ultrasonically treated, with 0.45 μm of filter membrane mistake
Filter, collects filtrate standby.
2. thick pure
Purification condition：Chromatographic column：Filler be SP high flow rate agarose microbeads Bio-sepFF, the strong sun of 50-160 μm of particle diameter
Ion exchange column, it is 2500mL that pillar loads volume.Initial buffer liquid mobile phase is：0.01mol/L pH=3.9-4.1 acetic acid-
Sodium acetate aqueous solution, elution flow is mutually A phases：The Acetic acid-sodium acetate solution that concentration is 0.01mol/L, pH value is 5.5-6.2, B
Phase：The Acetic acid-sodium acetate solution that concentration is 1.0mol/L, pH value is 5.5-6.2, flow velocity 25mL/min.Detection wavelength:215nm.
Gradient：0 to 110 minutes initial buffer liquid mobile phases 100%, 110 to 170 minutes initial buffer liquid mobile phases：Elution buffer
Mobile phase is by 70:30 to 65:35, it is within 170 to 340 minutes initial buffer liquid mobile phase：Elution buffer mobile phase is 65:35 is permanent
Stream.
Purge process：Cation exchange column is balanced each other to detector efflux pH=with the flowing of 100% initial buffer liquid
3.9-4.1, the invariable loading of electrical conductivity.Linear gradient elution, the peptide solution for collecting purpose peak value is stand-by.
3. desalination
Purification condition：Chromatographic column：Filler be SBC MCI GEI F type 30-50 μm reverse-phase chromatography filler F types 30-50 μm, post
It is 3000mL that son loads volume.Mobile phase A is：Ultra-pure water, Mobile phase B is：Chromatographic Pure Methanol.Flow velocity 50mL/min.Detection ripple
It is long：215nm.Gradient：0 to 100 minutes mobile phase As：Mobile phase B is by 100:0 to 90:15, it is within 100 to 200 minutes mobile phase A:
Mobile phase B is 90:15 constant currents.
Purge process：By polymer column with the ultrapure water balance of 100% mobile phase A well after loading.Linear gradient elution, collects
The peptide solution of purpose peak value, by the purpose peptide solution after desalination less than 40 DEG C of reduced pressure concentrations, is concentrated into about 50mg-100mg/mL
After freeze, that is, obtain the acetic acid type Argireline certified products pressed powder of purity 96%.
Embodiment 10
1. molten sample：100.0g synthesis in solid state gained Argireline is weighed, with 1000ml, pH=4,0.01mol/L acetic acid-acetic acid
Sodium water solution dissolves (concentration is about 100mg/mL), and with 1mol/L NaOH pH=3.9-4.1 is adjusted, ultrasonically treated, uses 0.45 μ
M membrane filtrations, collect filtrate standby.
2. thick pure
Purification condition：Chromatographic column：Filler be SP high flow rate agarose microbeads Bio-sepFF, the strong sun of 50-160 μm of particle diameter
Ion exchange column, it is 2500mL that pillar loads volume.Initial buffer liquid mobile phase is：0.01mol/L pH=3.9-4.1 acetic acid-
Sodium acetate aqueous solution, elution flow is mutually A phases：The Acetic acid-sodium acetate solution that concentration is 0.1mol/L, pH value is 5.5-6.2, B
Phase：The Acetic acid-sodium acetate solution that concentration is 1.0mol/L, pH value is 5.5-6.2, flow velocity 25mL/min.Detection wavelength：215nm.
Gradient：0 to 120 minutes initial buffer liquid mobile phases 100%, 120 to 180 minutes initial buffer liquid mobile phases：Elution buffer
Mobile phase is by 70:30 to 65:35, it is within 180 to 340 minutes initial buffer liquid mobile phase：Elution buffer mobile phase is 65:35 is permanent
Stream.
Purge process：Cation exchange column is balanced each other to detector efflux pH=with the flowing of 100% initial buffer liquid
3.9-4.1, the invariable loading of electrical conductivity.Linear gradient elution, the peptide solution for collecting purpose peak value is stand-by.
3. desalination
Purification condition：Chromatographic column：Filler be SBC MCI GEI F type 30-50 μm reverse-phase chromatography filler F types 30-50 μm, post
It is 3000mL that son loads volume.Mobile phase A is：Ultra-pure water, Mobile phase B is：Chromatographic Pure Methanol.Flow velocity 50mL/min.Detection ripple
It is long：215nm.Gradient：0 to 100 minutes mobile phase As：Mobile phase B is by 100:0 to 90:15, it is within 100 to 200 minutes mobile phase A：
Mobile phase B is 90:15 constant currents.
Purge process:By polymer column with the ultrapure water balance of 100% mobile phase A well after loading.Linear gradient elution, collects
The peptide solution of purpose peak value, by the purpose peptide solution after desalination less than 40 DEG C of reduced pressure concentrations, is concentrated into about 50mg-100mg/mL
After freeze, that is, obtain the acetic acid type Argireline certified products pressed powder of purity 95.8%.
The present invention is hereinbefore disclosed with preferred embodiment, but it should be understood by those skilled in the art that, these
Embodiment is only used for describing the present invention, and the scope that should not be construed as limiting the invention.It should be noted that every implement with these
The equivalent change of example and displacement, all should be set to be covered by scope of the presently claimed invention.Therefore, protection scope of the present invention
Should be defined by the scope defined in claims.
Claims (11)
1. a kind of purification process of Argireline, it is characterised in that include：
(1) dissolve：By Argireline dissolving crude product to be purified in Acetic acid-sodium acetate solution, filtrate is filtrated to get；
(2) it is thick pure：The filtrate loading that step (1) is obtained carries out gradient elution to strong cat ion exchange column with thick pure mobile phase,
Collect eluent；
(3) desalination：The eluent loading that step (2) is collected carries out gradient elution to inverted polymer post with desalination mobile phase,
Argireline solution is collected, reduced pressure concentration obtains Argireline with freeze-drying.
2. purification process according to claim 1, it is characterised in that in step (1), Argireline crude product to be purified and vinegar
The ratio of acid-SAS is (0.5g-100g):(20ml-1000ml), it is preferable that Argireline crude product to be purified and vinegar
The ratio of acid-SAS is (0.5g-10g):(20ml-100ml).
3. purification process according to claim 1, it is characterised in that it is 0.01- that the Acetic acid-sodium acetate solution is concentration
0.1mol/L, pH value are the Acetic acid-sodium acetate solution of 3.9-4.1.
4. the purification process according to any one of claim 1-3, it is characterised in that the thick pure mobile phase is by initial buffer
Liquid mobile phase and elution buffer flowing phase composition, wherein, the initial buffer liquid mobile phase is Acetic acid-sodium acetate solution, institute
It is the initial buffer liquid mobile phase for adding sodium chloride to state elution buffer mobile phase；Preferably, the initial buffer liquid mobile phase
It is that concentration is 0.01-0.1mol/L, the Acetic acid-sodium acetate solution that pH value is 3.9-4.1, the elution buffer mobile phase is to add
Enter the initial buffer liquid mobile phase of 0.5-1.5mol/L sodium chloride.
5. the purification process according to any one of claim 1-3, it is characterised in that the desalination mobile phase is by mobile phase A
Constitute with Mobile phase B, wherein, the mobile phase A is ultra-pure water, and the Mobile phase B is Chromatographic Pure Methanol.
6. the purification process according to claim 4 or 5, it is characterised in that in step (2), the thick pure eluent gradient
It is initial buffer liquid mobile phase:Elution buffer mobile phase is by 100:0 arrives (80-60):(20-40).
7. the purification process according to claim 4 or 5, it is characterised in that in step (3), the desalination eluent gradient
It is mobile phase A:Mobile phase B is by (100-95):(0-5) (90-80) is arrived:(10-20).
8. the purification process according to any one of claim 1-3, it is characterised in that the thick pure mobile phase is by initial buffer
Liquid mobile phase and elution buffer flowing phase composition, wherein, the initial buffer liquid mobile phase is Acetic acid-sodium acetate solution, institute
State the Acetic acid-sodium acetate buffer solution that elution buffer mobile phase is high concentration, high ph-values；Preferably, the initial buffer liquid stream
Dynamic phase is that concentration is 0.01-0.1mol/L, the Acetic acid-sodium acetate solution that pH value is 3.9-4.1, the elution buffer mobile phase
By A phases and B phase compositions, A phases are that concentration is 0.01-0.1mol/L, the Acetic acid-sodium acetate solution that pH value is 5.5-6.2, and B phases are
The Acetic acid-sodium acetate solution that concentration is 0.5-1.5mol/L, pH value is 5.5-6.2.
9. the purification process according to any one of claim 1-3, it is characterised in that the desalination mobile phase is by mobile phase A
Constitute with Mobile phase B, wherein, the mobile phase A is ultra-pure water, and the Mobile phase B is Chromatographic Pure Methanol.
10. purification process according to claim 8 or claim 9, it is characterised in that in step (2), the thick pure eluent gradient
It is initial buffer liquid mobile phase:Elution buffer mobile phase is by 100:0 arrives (80-60):(20-40).
11. purification process according to claim 8 or claim 9, it is characterised in that in step (3), the desalination eluent gradient
It is mobile phase A:Mobile phase B is by (100-95):(0-5) (90-80) is arrived:(5-10).
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| PCT/CN2017/075124 WO2018126524A1 (en) | 2017-01-04 | 2017-02-28 | Method for purifying areginine essence |
| AU2017100834A AU2017100834A4 (en) | 2017-01-04 | 2017-06-23 | Method for purifying argireline |
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| CN110426476A (en) * | 2019-08-15 | 2019-11-08 | 珠海伊斯佳科技股份有限公司 | The measuring method of Argireline content in a kind of cosmetics |
| CN115806591A (en) * | 2023-02-09 | 2023-03-17 | 杭州信海医药科技有限公司 | Purification method of high-purity vilacatide |
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| CN115500207A (en) * | 2022-10-20 | 2022-12-23 | 湖南口味王集团有限责任公司 | Areca nut powder, preparation method and application thereof, areca nut culture medium and application thereof |
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| CN103694316A (en) * | 2013-12-31 | 2014-04-02 | 杭州华津药业股份有限公司 | Preparation method of argireline |
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| CN103613655A (en) * | 2013-11-20 | 2014-03-05 | 陕西东大生化科技有限责任公司 | Method for low-cost purification of exenatide |
| CN103694316A (en) * | 2013-12-31 | 2014-04-02 | 杭州华津药业股份有限公司 | Preparation method of argireline |
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| CN110426476A (en) * | 2019-08-15 | 2019-11-08 | 珠海伊斯佳科技股份有限公司 | The measuring method of Argireline content in a kind of cosmetics |
| CN115806591A (en) * | 2023-02-09 | 2023-03-17 | 杭州信海医药科技有限公司 | Purification method of high-purity vilacatide |
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