CN102718841B - Method for purification of peptide biological material - Google Patents
Method for purification of peptide biological material Download PDFInfo
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- CN102718841B CN102718841B CN2012102143284A CN201210214328A CN102718841B CN 102718841 B CN102718841 B CN 102718841B CN 2012102143284 A CN2012102143284 A CN 2012102143284A CN 201210214328 A CN201210214328 A CN 201210214328A CN 102718841 B CN102718841 B CN 102718841B
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Abstract
The invention relates to a method for purification of a peptide biological material, which comprising the following steps of: dissolving the peptide biological material in trifluoroacetic acid, and filtering to obtain filtrate; taking cyano silane bonded silica gel as a chromatographic column of a stationary phase, diluting the filtrate of the peptide biological material with pure water, and taking a sample; taking aqueous solution of trifluoroacetic acid as Phase A and acetonitrile in the trifluoroacetic acid as Phase B, and performing linear gradient elution; taking methyl silane bonded silica gel as a stationary phase, and filling the obtained purified sample; taking aqueous solution of TFA (trifluoroacetic acid) as Phase A and acetonitrile as Phase B, and performing linear gradient elution; taking methyl silane bonded silica gel as a stationary phase, and filling the obtained purified sample; taking aqueous solution of ammonium acetate as Phase A1 and chromatographic pure acetonitrile as Phase B, and performing constant-gradient elution; taking aqueous solution of acetic acid as Phase A2 and chromatographic pure acetonitrile as Phase B, performing constant-gradient elution, and collecting target peak distillate; and drying to obtain fine peptide. The purification method is simple, the product purity is high, the yield is good, and the method can be industrialized easily.
Description
Technical field
The present invention relates to the purification process of peptide biomaterial, relate in particular to the purification process that is rich in R, A, the amino acid whose peptide biomaterial of D.
Background technology
The biomaterial that recent findings one class is comprised of the oligopeptides of spontaneous self assembly.The composition of these biomaterial scaffolds is that the complementary both sexes oligopeptides of oneself forms, their well-regulated repeating units: the amino-acid residue of positively charged (Methionin or arginine) and electronegative amino-acid residue (aspartic acid or L-glutamic acid) are separated by hydrophobic residue (L-Ala or leucine).The complementary both sexes oligopeptides of oneself comprises 50% charged residue, and take the ionic hydrophilic that replaces and the cycle of uncharged hydrophobic amino acid, repeats as feature.Typical example comprises RAD16-I, and its molecular structure is Ac-RADARADARADARADA-NH
2, RAD16-II, its molecular structure are Ac-RARADADARARADADA-NH
2RAD16-I has the spatial model of 4 (RADA), and RAD16-II only has the spatial model of 2 (RARADADA).Can be woven into the various geometric formats of relative thickness Deng the matrix scaffold of buoyancy (free-floating in solution neither sinks to also not rising to surface), or image-tape, or the picture lines, or slabbing.Peptide and salt concn,, together with the dimension of process instrumentation, determine geometry and the dimension of macroscopical matrix.Circle property dichroism spectroscope shows that having the peptide chain of typical cycle as described before based on the RAD model shows strong β lamella secondary structure in the aqueous solution.
The structure of the both sexes polypeptide of RAD and cell adhesion acceptor integrin RGD have similarity.Cell is adsorbed on based on RAD matrix being integrin dependence pattern, and based on the matrix sustenticular cell of RAD, adsorbs and growth.Research shows: RAD-16II is a kind of biomaterial scaffolds that can assist correct cytodifferentiation.
The peptide of this sequence is because having formed stronger β laminated structure, solvability in the aqueous solution is very poor, be difficult to lower prop during preparation on octadecylsilane base silica filler preparative column or butane group silanized silica gel filler preparative column, prepare the peak hangover very serious, purification ratio is more difficult.Yield is very low, with octadecylsilane commonly used or butyl silylation filler, prepares the very difficult industrialization production that is not easy to.
But the present invention proposes RAD-16I that a kind of purifying solid phase synthesis obtains and the purification process of RAD-16II, method is simple, and product purity is high and yield good and be easy to industrialization.
Summary of the invention
But proposed RAD-16I that a kind of purifying solid phase synthesis obtains and the purification process of RAD-16II, method is simple, and product purity is high and yield good and be easy to industrialization.
For achieving the above object, technical scheme provided by the invention comprises the following steps:
1) peptide biomaterial RAD-16I or RAD-16II adopt trifluoracetic acid to dissolve, and filter to get filtrate; The preferred pure trifluoracetic acid of described trifluoracetic acid.
2) chromatographic column take the cyanoalkysilane bonded silica gel as stationary phase, dilute loading with the filtrate of step 1) peptide biomaterial with pure water; Trifluoroacetic acid aqueous solution is the A phase, and volumetric concentration is that 0.05%-0.4% trifluoroacetic acetonitrile is the B phase, and linear gradient elution is collected purpose peak cut;
B phase gradient: 1%-30% B, flow rates 5ml/min-500ml/min.Dilution loading, the multiple of dilution are 0.5-1.5 times, preferred 1 times; The volumetric concentration of the mutually described trifluoroacetic acid aqueous solution of A is 0.1%-0.5%, is preferably 0.2%.
Because the hydrophobicity of peptide biomaterial RAD-16I or RAD-16II is strong, general silicagel column, such as the separating effect of octadecylsilane base silica filler is very undesirable, the applicant is surprised to find that, cyanoalkysilane bonded silica gel filler has desirable effect when separating for the peptide biomaterial.
The concentration of determining the B phase is the technical problem of a complexity, when the volumetric concentration of B item is too low, can not the Realization analysis purpose, when B item excessive concentration, acid large, stopping composition there is is the injury of increasing. in order to realize balance between the normal use of separating effect and packed column, need to make and selecting the concentration of B item. the applicant is surprised to find that, when B item volumetric concentration between 0.05%-0.4% the time, can realize purpose of the present invention, the stable use that also can keep packed column simultaneously, preferred volume concentration is 0.2%.
Gradient: 1%-30% B.In actually operating, graded can cause sample to be gone out too soon.Sample will be rushed out, and there is no separating effect.The speed range of graded: between minute one gradient of 1 minute three gradient to 5.
The preferred HPLC of chromatographic column, HPLC refers to high performance liquid chromatograph.
3) the methyl-monosilane bonded silica gel is stationary phase, with step 2) the purification of samples loading that obtains; Volumetric concentration is that 0.1%-0.2%TFA aqueous solution is the A phase, and volumetric concentration is that the acetonitrile of 0.1%-0.2%TFA is the B phase, and linear gradient elution is collected purpose peak cut;
B phase gradient: 10%-40%; TFA refers to trifluoracetic acid.
Peptide biomaterial RAD-16I or RAD-16II are difficult to separate at octadecylsilane base silica filler, perhaps go out the peak hangover, but can realize good separation on the methyl-monosilane based filler, go out peak and just can not trail.The resolution that the methyl-monosilane based filler is compared the cyanoalkysilane based filler is good, after peptide biomaterial RAD-16I or the separation of RAD-16II use cyanoalkysilane based filler, purity does not still reach requirement, again with the methyl-monosilane based filler, separate once, separating effect preferably can be arranged, also lower prop well, guarantee yield.
The preferred HPLC of chromatographic column, HPLC refers to high performance liquid chromatograph.
4) chromatographic column take the methyl-monosilane bonded silica gel as stationary phase, the purification of samples loading that step 3) is obtained; Take volumetric concentration as 0.1%-0.2%, pH value 5.0-7.0 ammonium acetate aqueous solution is the A1 phase, and trifluoroacetic acid aqueous solution is the B phase, and gradient is (1-10) %B+ (99-90) A1 Gradient elution 10-15min; And then take the 0.1%-0.2% aqueous acetic acid as the A2 phase, trifluoroacetic acid aqueous solution is the B phase, and gradient is (1-10) %B+ (99-90) A2 Gradient elution 10-15 minute, and sample, with 50%A2+50%B wash-out lower prop, is collected purpose peak cut; Dry smart peptide.
The preferred HPLC of chromatographic column, HPLC refers to high performance liquid chromatograph.
Above concentration is calculated with volumetric concentration.The concentration of moving phase and pH value are too low, and while turning salt, trifluoracetic acid is difficult to Ex-all, concentration is too high waste reagent.Be the balance that A1 can realize mutually turning salt efficiency and turn the salt cost so volumetric concentration is the 0.1%-0.2% ammonium acetate aqueous solution.The pH value is too high can damage preparative column, and sample may be separated out in preparative column simultaneously, and stops up preparative column, and ammonium acetate aqueous solution pH value 5.0-7.0 is desirable.
Wherein " degree of grade " is constant gradient volume implication.The parameter of flow velocity is 5ml/min-500ml/min.
With the desalting and purifying cut carry out vacuum rotary steam concentrated after, carry out lyophilize and namely obtain active sample.
Prior art is used the 18 alkyl silica gel filler, and peak is very undesired, serious hangover, and separating effect is very undesirable, and yield is extremely low.Can address the above problem well and with cyanoalkysilane based filler and methyl-monosilane based filler, unite use.
Simultaneously, peptide biomaterial RAD-16I or RAD-16II solvability are bad, reagent commonly used, and such as water, acetonitrile, methyl alcohol, Virahol, acetic acid, ammoniacal liquor etc., be difficult to dissolve.The present invention adopts trifluoracetic acid to dissolve, and realizes good solute effect; After having dissolved, thin up again during loading, produced good separation and purification effect.
Operation is simple and feasible for the method for purifying provided by the invention, purity reaches more than 98%, the purifying total recovery can reach more than 60%, reaches industrialized requirement.
The purifying scale comprises all size chromatographic column, and can linear amplification.Chromatographic column inner diameter d c is R (R=50 mm or other size), such as pillar internal diameter: 20mm, 50 mm, 100 mm etc.
Embodiment:
Embodiment one:
1. sample preparation: after with the pure TFA of 10ml is ultrasonic, sample being dissolved fully the thick peptide of 1.0g solid RAD-16II, use membrane filtration, collect filtrate for later use.
2. the first step HPLC purifying:
Purification condition: chromatographic column: the chromatographic column take the cyanoalkysilane bonded silica gel as stationary phase, pillar diameter and length are: 20 mm * 250 mm.Moving phase: A phase: the 0.2% trifluoroacetic acid liquid aqueous solution; The B phase: 0.1% trifluoroacetic acetonitrile, flow velocity: 19ml/min, gradient: 1% B-16% B, detect wavelength: 230 nm.Sample size is 1.0g.
Purge process: rinse chromatographic column well the rear use 99% A phase+1%B loading that balances each other with the acetonitrile more than 50%, applied sample amount is 1.0g, and during loading, sample filtrate is with one times of pure water dilution.Linear gradient elution 45min, collect the purpose peak, obtains purity greater than 80% above cut, the purpose peak cut of collection is no more than in water temperature under the condition of 35 ℃ and makes the second step purification of samples after the complete acetonitrile of vacuum rotary steam.
3. second step HPLC purifying:
Purification condition: chromatographic column: the chromatographic column take the methyl-monosilane bonded silica gel as stationary phase, pillar diameter and length are: 20 mm * 250 mm.Moving phase: A phase: the 0.1% trifluoroacetic aqueous solution is the A phase, and 0.1% trifluoroacetic acetonitrile is the B phase, gradient: 8% B-23% B, detect wavelength: 230 nm.
Purge process: chromatographic column is rinsed well after balance the first step purifying cut loading with the acetonitrile more than 50%.Linear gradient elution 45min, collect the purpose peak, obtains purity greater than 95% above cut, the purpose peak cut of collecting is no more than in water temperature after the complete acetonitrile of condition backspin of 35 ℃ to do the 3rd and go on foot the desalting and purifying sample.
4. the 3rd go on foot the HPLC desalting and purifying: chromatographic column: the chromatographic column take the methyl-monosilane bonded silica gel as stationary phase, pillar diameter and length are: 20 mm * 250 mm.Concentration 0.2%, the pH value is that the aqueous solution of 6.5 ammonium acetates is A 1 phase, trifluoroacetic acid aqueous solution is the B phase, gradient: 95% A+5% B balance 10 minutes, and then with concentration 0.1%, the pH value is the A2 phase for the 6.5HAc aqueous solution, and trifluoroacetic acid aqueous solution is the B phase, gradient: 95%A2+5%B balance 10 minutes, and then with 50%A2+50%B with peptide wash-out lower prop.Detect wavelength: 230 nm.
Collect the purpose peak, obtain purity greater than 95% above cut, the purpose peak cut of collecting is no more than under the condition of 35 ℃ vacuum rotary steam in water temperature and is concentrated into approximately and carries out lyophilize after 50 mg/ml, can obtain purity and be 95% RAD-16II essence peptide 710mg, the purifying total recovery is 71%.
Embodiment two:
1. sample preparation: after with the pure TFA of 10ml is ultrasonic, sample being dissolved fully the thick peptide of 1.0g solid RAD-16I, use membrane filtration, collect filtrate for later use.
2. the first step HPLC purifying:
Purification condition: chromatographic column: the chromatographic column take the cyanoalkysilane bonded silica gel as stationary phase, pillar diameter and length are: 20 mm * 250 mm.Moving phase: A phase: the 0.2% trifluoroacetic acid liquid aqueous solution; The B phase: 0.1% trifluoroacetic acetonitrile, flow velocity: 19ml/min, gradient: 1% B-16% B, detect wavelength: 230 nm.Sample size is 1.0g.
Purge process: chromatographic column is rinsed with the acetonitrile more than 50% well rear applied sample amount is 1.0g with 99%A phase+1%B loading that balances each other, during loading, sample filtrate is with one times of pure water dilution.Linear gradient elution 45min, collect the purpose peak, obtains purity greater than 80% above cut, the purpose peak cut of collection is no more than in water temperature under the condition of 35 ℃ and makes the second step purification of samples after the complete acetonitrile of vacuum rotary steam.
3. second step HPLC purifying:
Purification condition: chromatographic column: the chromatographic column take the methyl-monosilane bonded silica gel as stationary phase, pillar diameter and length are: 20 mm * 250 mm.Moving phase: A phase: the 0.1% trifluoroacetic aqueous solution is the A phase, and 0.1% trifluoroacetic acetonitrile is the B phase, gradient: 8% B-23% B, detect wavelength: 230 nm.
Purge process: chromatographic column is rinsed well after balance the first step purifying cut loading with the acetonitrile more than 50%.Linear gradient elution 45min, collect the purpose peak, obtains purity greater than 95% above cut, the purpose peak cut of collecting is no more than in water temperature after the complete acetonitrile of condition backspin of 35 ℃ to do the 3rd and go on foot the desalting and purifying sample.
4. the 3rd go on foot the HPLC desalting and purifying: chromatographic column: the chromatographic column take the methyl-monosilane bonded silica gel as stationary phase, pillar diameter and length are: 20 mm * 250 mm.Concentration 0.2%, the pH value is that the aqueous solution of 6.5 ammonium acetates is A 1 phase, trifluoroacetic acid aqueous solution is the B phase, gradient: 95% A+5% B balance 10 minutes, and then with concentration 0.1%, the pH value is the A2 phase for the 6.5HAc aqueous solution, and trifluoroacetic acid aqueous solution is the B phase, gradient: 95%A2+5%B balance 10 minutes, and then with 50%A2+50%B with peptide wash-out lower prop.Detect wavelength: 230 nm.
Collect the purpose peak, obtain purity greater than 95% above cut, the purpose peak cut of collecting is no more than under the condition of 35 ℃ vacuum rotary steam in water temperature and is concentrated into approximately and carries out lyophilize after 50 mg/ml, can obtain purity and be 95% RAD-16I essence peptide 720mg, the purifying total recovery is 72%.
Embodiment three:
1. sample preparation: after with the pure TFA of 10ml is ultrasonic, sample being dissolved fully the thick peptide of 1.5g solid RAD-16II, use membrane filtration, collect filtrate for later use.
2. the first step HPLC purifying:
Purification condition: chromatographic column: the chromatographic column take the cyanoalkysilane bonded silica gel as stationary phase, pillar diameter and length are: 20 mm * 250 mm.Moving phase: A phase: the 0.2% trifluoroacetic acid liquid aqueous solution; The B phase: 0.1% trifluoroacetic acetonitrile, flow velocity: 19ml/min, gradient: 1% B-16% B, detect wavelength: 230 nm.Sample size is 1.5g.
Purge process: chromatographic column is rinsed with the acetonitrile more than 50% well rear applied sample amount is 1.5g with 99%A phase+1%B loading that balances each other, during loading, sample filtrate is with one times of pure water dilution.Linear gradient elution 45min, collect the purpose peak, obtains purity greater than 80% above cut, the purpose peak cut of collection is no more than in water temperature under the condition of 35 ℃ and makes the second step purification of samples after the complete acetonitrile of vacuum rotary steam.
3. second step HPLC purifying:
Purification condition: chromatographic column: the chromatographic column take the methyl-monosilane bonded silica gel as stationary phase, pillar diameter and length are: 20 mm * 250 mm.Moving phase: A phase: the 0.1% trifluoroacetic aqueous solution is the A phase, and 0.1% trifluoroacetic acetonitrile is the B phase, gradient: 8% B-23% B, detect wavelength: 230 nm.
Purge process: chromatographic column is rinsed well after balance the first step purifying cut loading with the acetonitrile more than 50%.Linear gradient elution 45min, collect the purpose peak, obtains purity greater than 95% above cut, the purpose peak cut of collecting is no more than in water temperature after the complete acetonitrile of condition backspin of 35 ℃ to do the 3rd and go on foot the desalting and purifying sample.
4. the 3rd go on foot the HPLC desalting and purifying: chromatographic column: the chromatographic column take the methyl-monosilane bonded silica gel as stationary phase, pillar diameter and length are: 20 mm * 250 mm.Concentration 0.2%, the pH value is that the aqueous solution of 6.5 ammonium acetates is A 1 phase, trifluoroacetic acid aqueous solution is the B phase, gradient: 95% A+5% B balance 10 minutes, and then with concentration 0.1%, the pH value is the A2 phase for the 6.5HAc aqueous solution, and trifluoroacetic acid aqueous solution is the B phase, gradient: 95%A2+5%B balance 10 minutes, and then with 50%A2+50%B with peptide wash-out lower prop.Detect wavelength: 230 nm.
Collect the purpose peak, obtain purity greater than 95% above cut, the purpose peak cut of collecting is no more than under the condition of 35 ℃ vacuum rotary steam in water temperature and is concentrated into approximately and carries out lyophilize after 50 mg/ml, can obtain purity and be 95% RAD-16II essence peptide 923mg, the purifying total recovery is 61.5%.
Embodiment four:
1. sample preparation: after with the pure TFA of 30ml is ultrasonic, sample being dissolved fully the thick peptide of 4.5g solid RAD-16II, use membrane filtration, collect filtrate for later use.
2. the first step HPLC purifying:
Purification condition: chromatographic column: the chromatographic column take the cyanoalkysilane bonded silica gel as stationary phase, pillar diameter and length are: 50 mm * 250 mm.Moving phase: A phase: the 0.2% trifluoroacetic acid liquid aqueous solution; The B phase: 0.1% trifluoroacetic acetonitrile, flow velocity: 80ml/min, gradient: 1% B-16% B, detect wavelength: 230 nm.Sample size is 4.5g.
Purge process: chromatographic column is rinsed with the acetonitrile more than 50% well rear applied sample amount is 4.5g with 99%A phase+1%B loading that balances each other, during loading, sample filtrate is with one times of pure water dilution.Linear gradient elution 45min, collect the purpose peak, obtains purity greater than 80% above cut, the purpose peak cut of collection is no more than in water temperature under the condition of 35 ℃ and makes the second step purification of samples after the complete acetonitrile of vacuum rotary steam.
3. second step HPLC purifying:
Purification condition: chromatographic column: the chromatographic column take the methyl-monosilane bonded silica gel as stationary phase, pillar diameter and length are: 50 mm * 250 mm.Moving phase: A phase: the 0.1% trifluoroacetic aqueous solution is the A phase, and 0.1% trifluoroacetic acetonitrile is the B phase, gradient: 8% B-23% B, detect wavelength: 230 nm.
Purge process: chromatographic column is rinsed well after balance the first step purifying cut loading with the acetonitrile more than 50%.Linear gradient elution 45min, collect the purpose peak, obtains purity greater than 95% above cut, the purpose peak cut of collecting is no more than in water temperature after the complete acetonitrile of condition backspin of 35 ℃ to do the 3rd and go on foot the desalting and purifying sample.
4. the 3rd go on foot the HPLC desalting and purifying: chromatographic column: the chromatographic column take the methyl-monosilane bonded silica gel as stationary phase, pillar diameter and length are: 50 mm * 250 mm.Concentration 0.2%, the pH value is that the aqueous solution of 6.5 ammonium acetates is A 1 phase, trifluoroacetic acid aqueous solution is the B phase, gradient: 95% A+5% B balance 10 minutes, and then with concentration 0.1%, the pH value is the A2 phase for the 6.5HAc aqueous solution, and trifluoroacetic acid aqueous solution is the B phase, gradient: 95%A2+5%B balance 10 minutes, and then with 50%A2+50%B with peptide wash-out lower prop.Detect wavelength: 230 nm.
Collect the purpose peak, obtain purity greater than 95% above cut, the purpose peak cut of collecting is no more than under the condition of 35 ℃ vacuum rotary steam in water temperature and is concentrated into approximately and carries out lyophilize after 50 mg/ml, can obtain purity and be 95% RAD-16II essence peptide 2.9g, the purifying total recovery is 64.4%.
Embodiment five:
1. sample preparation: after with the pure TFA of 35ml is ultrasonic, sample being dissolved fully the thick peptide of 5.1g solid RAD-16II, use membrane filtration, collect filtrate for later use.
2. the first step HPLC purifying:
Purification condition: chromatographic column: the chromatographic column take the cyanoalkysilane bonded silica gel as stationary phase, pillar diameter and length are: 50 mm * 250 mm.Moving phase: A phase: the 0.2% trifluoroacetic acid liquid aqueous solution; The B phase: 0.1% trifluoroacetic acetonitrile, flow velocity: 80ml/min, gradient: 1% B-16% B, detect wavelength: 230 nm.Sample size is 4.5g.
Purge process: chromatographic column is rinsed with the acetonitrile more than 50% well rear applied sample amount is 4.5g with 99%A phase+1%B loading that balances each other, during loading, sample filtrate is with one times of pure water dilution.Linear gradient elution 45min, collect the purpose peak, obtains purity greater than 80% above cut, the purpose peak cut of collection is no more than in water temperature under the condition of 35 ℃ and makes the second step purification of samples after the complete acetonitrile of vacuum rotary steam.
3. second step HPLC purifying:
Purification condition: chromatographic column: the chromatographic column take the methyl-monosilane bonded silica gel as stationary phase, pillar diameter and length are: 50 mm * 250 mm.Moving phase: A phase: the 0.1% trifluoroacetic aqueous solution is the A phase, and 0.1% trifluoroacetic acetonitrile is the B phase, gradient: 8% B-23% B, detect wavelength: 230 nm.
Purge process: chromatographic column is rinsed well after balance the first step purifying cut loading with the acetonitrile more than 50%.Linear gradient elution 45min, collect the purpose peak, obtains purity greater than 95% above cut, the purpose peak cut of collecting is no more than in water temperature after the complete acetonitrile of condition backspin of 35 ℃ to do the 3rd and go on foot the desalting and purifying sample.
4. the 3rd go on foot the HPLC desalting and purifying: chromatographic column: the chromatographic column take the methyl-monosilane bonded silica gel as stationary phase, pillar diameter and length are: 50 mm * 250 mm.Concentration 0.2%, the pH value is that the aqueous solution of 6.5 ammonium acetates is A 1 phase, trifluoroacetic acid aqueous solution is the B phase, gradient: 95% A+5% B balance 10 minutes, and then with concentration 0.1%, the pH value is the A2 phase for the 6.5HAc aqueous solution, and trifluoroacetic acid aqueous solution is the B phase, gradient: 95%A2+5%B balance 10 minutes, and then with 50%A2+50%B with peptide wash-out lower prop.Detect wavelength: 230 nm.
Collect the purpose peak, obtain purity greater than 95% above cut, the purpose peak cut of collecting is no more than under the condition of 35 ℃ vacuum rotary steam in water temperature and is concentrated into approximately and carries out lyophilize after 50 mg/ml, can obtain purity and be 95% RAD-16II essence peptide 2.9g, the purifying total recovery is 64.4%.
Embodiment six:
1. sample preparation: after with the pure TFA of 35ml is ultrasonic, sample being dissolved fully the thick peptide of 5.0g solid RAD-16I, use membrane filtration, collect filtrate for later use.
2. the first step HPLC purifying:
Purification condition: chromatographic column: the chromatographic column take the cyanoalkysilane bonded silica gel as stationary phase, pillar diameter and length are: 50 mm * 250 mm.Moving phase: A phase: the 0.2% trifluoroacetic acid liquid aqueous solution; The B phase: 0.1% trifluoroacetic acetonitrile, flow velocity: 80ml/min, gradient: 1% B-16% B, detect wavelength: 230 nm.Sample size is 4.5g.
Purge process: chromatographic column is rinsed with the acetonitrile more than 50% well rear applied sample amount is 5.0g with 99%A phase+1%B loading that balances each other, during loading, sample filtrate is with one times of pure water dilution.Linear gradient elution 45min, collect the purpose peak, obtains purity greater than 80% above cut, the purpose peak cut of collection is no more than in water temperature under the condition of 35 ℃ and makes the second step purification of samples after the complete acetonitrile of vacuum rotary steam.
3. second step HPLC purifying:
Purification condition: chromatographic column: the chromatographic column take the methyl-monosilane bonded silica gel as stationary phase, pillar diameter and length are: 50 mm * 250 mm.Moving phase: A phase: the 0.1% trifluoroacetic aqueous solution is the A phase, and 0.1% trifluoroacetic acetonitrile is the B phase, gradient: 8% B-23% B, detect wavelength: 230 nm.
Purge process: chromatographic column is rinsed well after balance the first step purifying cut loading with the acetonitrile more than 50%.Linear gradient elution 45min, collect the purpose peak, obtains purity greater than 95% above cut, the purpose peak cut of collecting is no more than in water temperature after the complete acetonitrile of condition backspin of 35 ℃ to do the 3rd and go on foot the desalting and purifying sample.
4. the 3rd go on foot the HPLC desalting and purifying: chromatographic column: the chromatographic column take the methyl-monosilane bonded silica gel as stationary phase, pillar diameter and length are: 50 mm * 250 mm.Concentration 0.2%, the pH value is that the aqueous solution of 6.5 ammonium acetates is A 1 phase, trifluoroacetic acid aqueous solution is the B phase, gradient: 95% A+5% B balance 10 minutes, and then with concentration 0.1%, the pH value is the A2 phase for the 6.5HAc aqueous solution, and trifluoroacetic acid aqueous solution is the B phase, gradient: 95%A2+5%B balance 10 minutes, and then with 50%A2+50%B with peptide wash-out lower prop.Detect wavelength: 230 nm.
Collect the purpose peak, obtain purity greater than 95% above cut, the purpose peak cut of collecting is no more than under the condition of 35 ℃ vacuum rotary steam in water temperature and is concentrated into approximately and carries out lyophilize after 50 mg/ml, can obtain purity and be 95% RAD-16I essence peptide 3.1g, the purifying total recovery is 62%.
Embodiment seven:
1. sample preparation: after with the pure TFA of 50ml is ultrasonic, sample being dissolved fully the thick peptide of 10.0g solid RAD-16I, use membrane filtration, collect filtrate for later use.
2. the first step HPLC purifying:
Purification condition: chromatographic column: the chromatographic column take the cyanoalkysilane bonded silica gel as stationary phase, pillar diameter and length are: 100 mm * 250 mm.Moving phase: A phase: the 0.2% trifluoroacetic acid liquid aqueous solution; The B phase: 0.1% trifluoroacetic acetonitrile, flow velocity: 220ml/min, gradient: 1% B-16% B, detect wavelength: 230 nm.Sample size is 10.0g.
Purge process: chromatographic column is rinsed with the acetonitrile more than 50% well rear applied sample amount is 10.0g with 99%A phase+1%B loading that balances each other, during loading, sample filtrate is with one times of pure water dilution.Linear gradient elution 45min, collect the purpose peak, obtains purity greater than 80% above cut, the purpose peak cut of collection is no more than in water temperature under the condition of 35 ℃ and makes the second step purification of samples after the complete acetonitrile of vacuum rotary steam.
3. second step HPLC purifying:
Purification condition: chromatographic column: the chromatographic column take the methyl-monosilane bonded silica gel as stationary phase, pillar diameter and length are: 100 mm * 250 mm.Moving phase: A phase: the 0.1% trifluoroacetic aqueous solution is the A phase, and 0.1% trifluoroacetic acetonitrile is the B phase, gradient: 8% B-23% B, detect wavelength: 230 nm.
Purge process: chromatographic column is rinsed well after balance the first step purifying cut loading with the acetonitrile more than 50%.Linear gradient elution 45min, collect the purpose peak, obtains purity greater than 95% above cut, the purpose peak cut of collecting is no more than in water temperature after the complete acetonitrile of condition backspin of 35 ℃ to do the 3rd and go on foot the desalting and purifying sample.
4. the 3rd go on foot the HPLC desalting and purifying: chromatographic column: the chromatographic column take the methyl-monosilane bonded silica gel as stationary phase, pillar diameter and length are: 100 mm * 250 mm.Concentration 0.2%, the pH value is that the aqueous solution of 6.5 ammonium acetates is A 1 phase, trifluoroacetic acid aqueous solution is the B phase, gradient: 95% A+5% B balance 10 minutes, and then with concentration 0.1%, the pH value is the A2 phase for the 6.5HAc aqueous solution, and trifluoroacetic acid aqueous solution is the B phase, gradient: 95%A2+5%B balance 10 minutes, and then with 50%A2+50%B with peptide wash-out lower prop.Detect wavelength: 230 nm.
Collect the purpose peak, obtain purity greater than 95% above cut, the purpose peak cut of collecting is no more than under the condition of 35 ℃ vacuum rotary steam in water temperature and is concentrated into approximately and carries out lyophilize after 50 mg/ml, can obtain purity and be 95% RAD-16I essence peptide 6.3g, the purifying total recovery is 63%.
Embodiment eight:
1. sample preparation: after with the pure TFA of 55ml is ultrasonic, sample being dissolved fully the thick peptide of 11.0g solid RAD-16II, use membrane filtration, collect filtrate for later use.
2. the first step HPLC purifying:
Purification condition: chromatographic column: the chromatographic column take the cyanoalkysilane bonded silica gel as stationary phase, pillar diameter and length are: 100 mm * 250 mm.Moving phase: A phase: the 0.2% trifluoroacetic acid liquid aqueous solution; The B phase: 0.1% trifluoroacetic acetonitrile, flow velocity: 80ml/min, gradient: 1% B-16% B, detect wavelength: 230 nm.Sample size is 11.0g.
Purge process: chromatographic column is rinsed with the acetonitrile more than 50% well rear applied sample amount is 11.0g with 99%A phase+1%B loading that balances each other, during loading, sample filtrate is with one times of pure water dilution.Linear gradient elution 45min, collect the purpose peak, obtains purity greater than 80% above cut, the purpose peak cut of collection is no more than in water temperature under the condition of 35 ℃ and makes the second step purification of samples after the complete acetonitrile of vacuum rotary steam.
3. second step HPLC purifying:
Purification condition: chromatographic column: the chromatographic column take the methyl-monosilane bonded silica gel as stationary phase, pillar diameter and length are: 100 mm * 250 mm.Moving phase: A phase: the 0.1% trifluoroacetic aqueous solution is the A phase, and 0.1% trifluoroacetic acetonitrile is the B phase, gradient: 8% B-23% B, detect wavelength: 230 nm.
Purge process: chromatographic column is rinsed well after balance the first step purifying cut loading with the acetonitrile more than 50%.Linear gradient elution 45min, collect the purpose peak, obtains purity greater than 95% above cut, the purpose peak cut of collecting is no more than in water temperature after the complete acetonitrile of condition backspin of 35 ℃ to do the 3rd and go on foot the desalting and purifying sample.
4. the 3rd go on foot the HPLC desalting and purifying: chromatographic column: the chromatographic column take the methyl-monosilane bonded silica gel as stationary phase, pillar diameter and length are: 100 mm * 250 mm.Concentration 0.2%, the pH value is that the aqueous solution of 6.5 ammonium acetates is A 1 phase, trifluoroacetic acid aqueous solution is the B phase, gradient: 95% A+5% B balance 10 minutes, and then with concentration 0.1%, the pH value is the A2 phase for the 6.5HAc aqueous solution, and trifluoroacetic acid aqueous solution is the B phase, gradient: 95%A2+5%B balance 10 minutes, and then with 50%A2+50%B with peptide wash-out lower prop.Detect wavelength: 230 nm.
Collect the purpose peak, obtain purity greater than 95% above cut, the purpose peak cut of collecting is no more than under the condition of 35 ℃ vacuum rotary steam in water temperature and is concentrated into approximately and carries out lyophilize after 50 mg/ml, can obtain purity and be 95% RAD-16II essence peptide 7.1g, the purifying total recovery is 64.5%.
Claims (2)
1. the purification process of a peptide biomaterial, described peptide biomaterial is RAD-16I or RAD-16II, the method comprises the following steps:
1) peptide biomaterial RAD-16I or RAD-16II adopt trifluoracetic acid to dissolve, and filter to get filtrate;
2) chromatographic column take the cyanoalkysilane bonded silica gel as stationary phase, dilute loading with the filtrate of step 1) peptide biomaterial with pure water; Adopting 0.2% trifluoroacetic acid aqueous solution is the A phase,, take volumetric concentration as 0.1% trifluoroacetic acetonitrile as the B phase,, take the B phase gradient as the 1%-16% linear gradient elution, collects purpose peak cut;
3) take the methyl-monosilane bonded silica gel as stationary phase, with step 2) the purification of samples loading that obtains; Volumetric concentration is that 0.1%-0.2%TFA aqueous solution is the A phase, and volumetric concentration is that the acetonitrile of 0.1%-0.2%TFA is the B phase,, take the B phase gradient as the 8%-23% linear gradient elution, collects purpose peak cut;
4) chromatographic column take the methyl-monosilane bonded silica gel as stationary phase, the purification of samples loading that step 3) is obtained; Take volumetric concentration as 0.1%-0.2%, pH value 5.0-7.0 ammonium acetate aqueous solution is the A1 phase, and trifluoroacetic acid aqueous solution is the B phase, and gradient is (1-10) %B+ (99-90) %A1 Gradient elution 10-15min; And then take the 0.1%-0.2% aqueous acetic acid as the A2 phase, trifluoroacetic acid aqueous solution is the B phase, and gradient is (1-10) %B+ (99-90) %A2 Gradient elution 10-15 minute, with 50%A2+50%B,, with sample wash-out lower prop, collects purpose peak cut; Dry smart peptide.
2. purification process as claimed in claim 1, it is characterized in that: described step 2), step 1) peptide biomaterial is dissolved with pure trifluoracetic acid, the filtrate of gained is diluted loading with pure water, the multiple of dilution be 0.5-5 doubly.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002062961A2 (en) * | 2001-02-06 | 2002-08-15 | Massachusetts Institute Of Technology | Peptide scaffold encapsulation of tissue cells and uses thereof |
| WO2008063418A2 (en) * | 2006-11-17 | 2008-05-29 | Abbott Cardiovascular Systems Inc. | Modified two-component gelation systems, methods of use and methods manufacture |
| CN101525382A (en) * | 2009-04-21 | 2009-09-09 | 深圳市翰宇药业有限公司 | Method of purifying pramlintide |
| CN101817873A (en) * | 2010-03-11 | 2010-09-01 | 复旦大学 | Cell adhesion promoting polypeptide and preparation method thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002062961A2 (en) * | 2001-02-06 | 2002-08-15 | Massachusetts Institute Of Technology | Peptide scaffold encapsulation of tissue cells and uses thereof |
| WO2008063418A2 (en) * | 2006-11-17 | 2008-05-29 | Abbott Cardiovascular Systems Inc. | Modified two-component gelation systems, methods of use and methods manufacture |
| CN101525382A (en) * | 2009-04-21 | 2009-09-09 | 深圳市翰宇药业有限公司 | Method of purifying pramlintide |
| CN101817873A (en) * | 2010-03-11 | 2010-09-01 | 复旦大学 | Cell adhesion promoting polypeptide and preparation method thereof |
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| RobertaGambaretto等.Self-AssemblingPeptides:Sequence Secondary Structure in Solution and Film Formation.《Biopolymers》.2008 |
| Self-Assembling Peptides: Sequence, Secondary Structure in Solution and Film Formation;Roberta Gambaretto等;《Biopolymers》;20080602;第89卷(第11期);906-915 * |
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