CN102718841A - Method for purification of peptide biological material - Google Patents
Method for purification of peptide biological material Download PDFInfo
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- CN102718841A CN102718841A CN2012102143284A CN201210214328A CN102718841A CN 102718841 A CN102718841 A CN 102718841A CN 2012102143284 A CN2012102143284 A CN 2012102143284A CN 201210214328 A CN201210214328 A CN 201210214328A CN 102718841 A CN102718841 A CN 102718841A
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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
One type of biomaterial of forming by the oligopeptides of spontaneous oneself's assembling of recent findings.The composition of these biomaterial scaffolds is that self-complementary both sexes oligopeptides is formed, their well-regulated repeating units: the amino-acid residue of positively charged (Methionin or l-arginine) and electronegative amino-acid residue (aspartic acid or L-glutamic acid) are separated by hydrophobic residue (L-Ala or leucine).Oneself's complementary both sexes oligopeptides comprises 50% charged residue, and to repeat with cycle of alternative ionic hydrophilic property and uncharged hydrophobic amino acid be characteristic.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 has only the spatial model of 2 (RARADADA).Matrix scaffold Deng buoyancy (free-floating in solution neither sinks to also not rising to the surface) can be woven into the various geometric formats of relative heavy-gravity, or image-tape, or the picture lines, or slabbing.Peptide and salt concn together with the dimension of process instrumentation, determine the geometry and the dimension of macroscopical matrix.Circle property dichroism spectroscope shows to have like the peptide chain of the typical cycle property of preceding description 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 being to integrate plain dependence pattern on the RAD matrix, and based on absorption of matrix sustenticular cell and the growth of RAD.Research shows: RAD-16II is a kind of biomaterial scaffolds that can assist correct cytodifferentiation.
The peptide of this sequence is because of having formed stronger β laminated structure; Solvability in the aqueous solution is very poor; On octadecylsilane base silica filler preparative column or butane group silanized silica gel filler preparative column, be difficult to lower prop during preparation, the hangover of preparation peak is very serious, and purification ratio is difficulty.Yield is very low, and preparing very with octadecylsilane commonly used or butyl siloyl group filler, difficulty is not easy to industrialization production.
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 height and yield are 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 height and yield are good and be easy to industrialization.
For realizing above-mentioned purpose, technical scheme provided by the invention may further comprise the steps:
1) peptide biomaterial RAD-16I or RAD-16II adopt the trifluoracetic acid dissolving, cross and filter filtrating; The preferred pure trifluoracetic acid of described trifluoracetic acid.
2) be the chromatographic column of stationary phase with cyanic acid silane group silica gel, the filtrating of step 1) peptide biomaterial is gone up appearance with the pure water dilution; 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.Appearance in the dilution, the multiple of dilution are 0.5-1.5 times, preferred 1 times; The volumetric concentration of the mutually said 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; Separating effect such as octadecylsilane base silica filler is very undesirable, and the applicant is surprised to find that cyanic acid silane group silica filler has the ideal effect for the peptide biomaterial when separating.
The concentration of confirming the B phase is a complicated technology problem, crosses when low when the volumetric concentration of B item, can not realize analysis purposes; When B item excessive concentration; Acid big, stopping composition there is the injury of increasing. in order to realize balance between the normal use of separating effect and packed column, need make the concentration of B item and selecting. the applicant is surprised to find that; When B item volumetric concentration between 0.05%-0.4% the time; Can realize the object of the invention, the stable use that also can keep packed column simultaneously, preferred volume concentration is 0.2%.
Gradient: 1%-30% B.Graded can cause sample to be gone out too soon in the actually operating.Sample will be rushed out, and does not have 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 a stationary phase, with step 2) appearance on the purification of samples 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 on the methyl-monosilane based filler, can realize good separation, go out the peak and just can not trail.The separating size that the methyl-monosilane based filler is compared cyanic acid siloyl group filler is good; After peptide biomaterial RAD-16I or RAD-16II separate with cyanic acid siloyl group filler; Purity does not still reach requirement, separates once with the methyl-monosilane based filler once more, and separating effect preferably can be arranged; Also lower prop well guarantees yield.
The preferred HPLC of chromatographic column, HPLC refers to high performance liquid chromatograph.
4) with the methyl-monosilane bonded silica gel be the chromatographic column of stationary phase, appearance on the purification of samples that step 3) is obtained; With the volumetric concentration is 0.1%-0.2%, and 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 constant gradient wash-out 10-15min; And then be the A2 phase with the 0.1%-0.2% aqueous acetic acid, trifluoroacetic acid aqueous solution is the B phase, gradient is (1-10) %B+ (99-90) A2 constant gradient wash-out 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 when changeing salt, trifluoracetic acid is difficult to Ex-all, concentration is too high waste reagent.So volumetric concentration is that A1 can realize changeing salt efficient and the balance of changeing the salt cost mutually for the 0.1%-0.2% ammonium acetate aqueous solution.The pH value is too high can damage preparative column, and sample possibly 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.
The desalting and purifying cut is carried out carrying out lyophilize and promptly getting active sample after vacuum rotary steam concentrates.
Prior art is used the 18 alkyl silica gel filler, and the peak is very undesired, serious hangover, and separating effect is very undesirable, and yield is extremely low.Can address the above problem well and unite use with cyanic acid siloyl group filler and methyl-monosilane based filler.
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. are difficult to dissolving.The present invention adopts the trifluoracetic acid dissolving, realizes good solute effect; After having dissolved, in last when appearance, be thin up again, has 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 sizes), for example 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: with cyanic acid silane group silica gel is the chromatographic column of stationary phase, and 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 back with the acetonitrile more than 50% and balance each other with 99% A phase+1%B and go up appearance, applied sample amount is 1.0g, and sample is filtrated and diluted one times with pure water during last appearance.Linear gradient elution 45min collects the purpose peak, obtains purity greater than 80% above cut, the purpose peak cut of collecting is no more than in water temperature do second behind the intact acetonitrile of vacuum rotary steam under 35 ℃ the condition and go on foot purification of samples.
3. second go on foot the HPLC purifying:
Purification condition: chromatographic column: with the methyl-monosilane bonded silica gel is the chromatographic column of stationary phase, and 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 the balance appearance on the first step purifying cut with the acetonitrile more than 50%.Linear gradient elution 45min collects the purpose peak, obtains purity greater than 95% above cut, the purpose peak cut of collecting is no more than in water temperature do the 3rd behind 35 ℃ the intact acetonitrile of condition underspin and go on foot the desalting and purifying sample.
4. the 3rd go on foot the HPLC desalting and purifying: chromatographic column: with the methyl-monosilane bonded silica gel is the chromatographic column of stationary phase, and pillar diameter and length are: 20 mm * 250 mm.Concentration 0.2%, pH value are that the aqueous solution of 6.5 ammonium acetates is A 1 phase, and 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 in water temperature carries out lyophilize after vacuum rotary steam is concentrated into about 50 mg/ml under 35 ℃ the condition, can obtain purity and be 95% the smart peptide 710mg of RAD-16II, 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: with cyanic acid silane group silica gel is the chromatographic column of stationary phase, and 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 back with the acetonitrile more than 50% and balance each other with 99%A phase+1%B and go up appearance, applied sample amount is 1.0g, and sample filtrating is diluted one times with pure water during last appearance.Linear gradient elution 45min collects the purpose peak, obtains purity greater than 80% above cut, the purpose peak cut of collecting is no more than in water temperature do second behind the intact acetonitrile of vacuum rotary steam under 35 ℃ the condition and go on foot purification of samples.
3. second go on foot the HPLC purifying:
Purification condition: chromatographic column: with the methyl-monosilane bonded silica gel is the chromatographic column of stationary phase, and 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 the balance appearance on the first step purifying cut with the acetonitrile more than 50%.Linear gradient elution 45min collects the purpose peak, obtains purity greater than 95% above cut, the purpose peak cut of collecting is no more than in water temperature do the 3rd behind 35 ℃ the intact acetonitrile of condition underspin and go on foot the desalting and purifying sample.
4. the 3rd go on foot the HPLC desalting and purifying: chromatographic column: with the methyl-monosilane bonded silica gel is the chromatographic column of stationary phase, and pillar diameter and length are: 20 mm * 250 mm.Concentration 0.2%, pH value are that the aqueous solution of 6.5 ammonium acetates is A 1 phase, and 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 in water temperature carries out lyophilize after vacuum rotary steam is concentrated into about 50 mg/ml under 35 ℃ the condition, can obtain purity and be 95% the smart peptide 720mg of RAD-16I, 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: with cyanic acid silane group silica gel is the chromatographic column of stationary phase, and 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: rinse chromatographic column well back with the acetonitrile more than 50% and balance each other with 99%A phase+1%B and go up appearance, applied sample amount is 1.5g, and sample filtrating is diluted one times with pure water during last appearance.Linear gradient elution 45min collects the purpose peak, obtains purity greater than 80% above cut, the purpose peak cut of collecting is no more than in water temperature do second behind the intact acetonitrile of vacuum rotary steam under 35 ℃ the condition and go on foot purification of samples.
3. second go on foot the HPLC purifying:
Purification condition: chromatographic column: with the methyl-monosilane bonded silica gel is the chromatographic column of stationary phase, and 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 the balance appearance on the first step purifying cut with the acetonitrile more than 50%.Linear gradient elution 45min collects the purpose peak, obtains purity greater than 95% above cut, the purpose peak cut of collecting is no more than in water temperature do the 3rd behind 35 ℃ the intact acetonitrile of condition underspin and go on foot the desalting and purifying sample.
4. the 3rd go on foot the HPLC desalting and purifying: chromatographic column: with the methyl-monosilane bonded silica gel is the chromatographic column of stationary phase, and pillar diameter and length are: 20 mm * 250 mm.Concentration 0.2%, pH value are that the aqueous solution of 6.5 ammonium acetates is A 1 phase, and 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 in water temperature carries out lyophilize after vacuum rotary steam is concentrated into about 50 mg/ml under 35 ℃ the condition, can obtain purity and be 95% the smart peptide 923mg of RAD-16II, 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: with cyanic acid silane group silica gel is the chromatographic column of stationary phase, and 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: rinse chromatographic column well back with the acetonitrile more than 50% and balance each other with 99%A phase+1%B and go up appearance, applied sample amount is 4.5g, and sample filtrating is diluted one times with pure water during last appearance.Linear gradient elution 45min collects the purpose peak, obtains purity greater than 80% above cut, the purpose peak cut of collecting is no more than in water temperature do second behind the intact acetonitrile of vacuum rotary steam under 35 ℃ the condition and go on foot purification of samples.
3. second go on foot the HPLC purifying:
Purification condition: chromatographic column: with the methyl-monosilane bonded silica gel is the chromatographic column of stationary phase, and 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 the balance appearance on the first step purifying cut with the acetonitrile more than 50%.Linear gradient elution 45min collects the purpose peak, obtains purity greater than 95% above cut, the purpose peak cut of collecting is no more than in water temperature do the 3rd behind 35 ℃ the intact acetonitrile of condition underspin and go on foot the desalting and purifying sample.
4. the 3rd go on foot the HPLC desalting and purifying: chromatographic column: with the methyl-monosilane bonded silica gel is the chromatographic column of stationary phase, and pillar diameter and length are: 50 mm * 250 mm.Concentration 0.2%, pH value are that the aqueous solution of 6.5 ammonium acetates is A 1 phase, and 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 in water temperature carries out lyophilize after vacuum rotary steam is concentrated into about 50 mg/ml under 35 ℃ the condition, can obtain purity and be 95% the smart peptide 2.9g of RAD-16II, 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: with cyanic acid silane group silica gel is the chromatographic column of stationary phase, and 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: rinse chromatographic column well back with the acetonitrile more than 50% and balance each other with 99%A phase+1%B and go up appearance, applied sample amount is 4.5g, and sample filtrating is diluted one times with pure water during last appearance.Linear gradient elution 45min collects the purpose peak, obtains purity greater than 80% above cut, the purpose peak cut of collecting is no more than in water temperature do second behind the intact acetonitrile of vacuum rotary steam under 35 ℃ the condition and go on foot purification of samples.
3. second go on foot the HPLC purifying:
Purification condition: chromatographic column: with the methyl-monosilane bonded silica gel is the chromatographic column of stationary phase, and 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 the balance appearance on the first step purifying cut with the acetonitrile more than 50%.Linear gradient elution 45min collects the purpose peak, obtains purity greater than 95% above cut, the purpose peak cut of collecting is no more than in water temperature do the 3rd behind 35 ℃ the intact acetonitrile of condition underspin and go on foot the desalting and purifying sample.
4. the 3rd go on foot the HPLC desalting and purifying: chromatographic column: with the methyl-monosilane bonded silica gel is the chromatographic column of stationary phase, and pillar diameter and length are: 50 mm * 250 mm.Concentration 0.2%, pH value are that the aqueous solution of 6.5 ammonium acetates is A 1 phase, and 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 in water temperature carries out lyophilize after vacuum rotary steam is concentrated into about 50 mg/ml under 35 ℃ the condition, can obtain purity and be 95% the smart peptide 2.9g of RAD-16II, 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: with cyanic acid silane group silica gel is the chromatographic column of stationary phase, and 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: rinse chromatographic column well back with the acetonitrile more than 50% and balance each other with 99%A phase+1%B and go up appearance, applied sample amount is 5.0g, and sample filtrating is diluted one times with pure water during last appearance.Linear gradient elution 45min collects the purpose peak, obtains purity greater than 80% above cut, the purpose peak cut of collecting is no more than in water temperature do second behind the intact acetonitrile of vacuum rotary steam under 35 ℃ the condition and go on foot purification of samples.
3. second go on foot the HPLC purifying:
Purification condition: chromatographic column: with the methyl-monosilane bonded silica gel is the chromatographic column of stationary phase, and 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 the balance appearance on the first step purifying cut with the acetonitrile more than 50%.Linear gradient elution 45min collects the purpose peak, obtains purity greater than 95% above cut, the purpose peak cut of collecting is no more than in water temperature do the 3rd behind 35 ℃ the intact acetonitrile of condition underspin and go on foot the desalting and purifying sample.
4. the 3rd go on foot the HPLC desalting and purifying: chromatographic column: with the methyl-monosilane bonded silica gel is the chromatographic column of stationary phase, and pillar diameter and length are: 50 mm * 250 mm.Concentration 0.2%, pH value are that the aqueous solution of 6.5 ammonium acetates is A 1 phase, and 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 in water temperature carries out lyophilize after vacuum rotary steam is concentrated into about 50 mg/ml under 35 ℃ the condition, can obtain purity and be 95% the smart peptide 3.1g of RAD-16I, 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: with cyanic acid silane group silica gel is the chromatographic column of stationary phase, and 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: rinse chromatographic column well back with the acetonitrile more than 50% and balance each other with 99%A phase+1%B and go up appearance, applied sample amount is 10.0g, and sample filtrating is diluted one times with pure water during last appearance.Linear gradient elution 45min collects the purpose peak, obtains purity greater than 80% above cut, the purpose peak cut of collecting is no more than in water temperature do second behind the intact acetonitrile of vacuum rotary steam under 35 ℃ the condition and go on foot purification of samples.
3. second go on foot the HPLC purifying:
Purification condition: chromatographic column: with the methyl-monosilane bonded silica gel is the chromatographic column of stationary phase, and 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 the balance appearance on the first step purifying cut with the acetonitrile more than 50%.Linear gradient elution 45min collects the purpose peak, obtains purity greater than 95% above cut, the purpose peak cut of collecting is no more than in water temperature do the 3rd behind 35 ℃ the intact acetonitrile of condition underspin and go on foot the desalting and purifying sample.
4. the 3rd go on foot the HPLC desalting and purifying: chromatographic column: with the methyl-monosilane bonded silica gel is the chromatographic column of stationary phase, and pillar diameter and length are: 100 mm * 250 mm.Concentration 0.2%, pH value are that the aqueous solution of 6.5 ammonium acetates is A 1 phase, and 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 in water temperature carries out lyophilize after vacuum rotary steam is concentrated into about 50 mg/ml under 35 ℃ the condition, can obtain purity and be 95% the smart peptide 6.3g of RAD-16I, 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: with cyanic acid silane group silica gel is the chromatographic column of stationary phase, and 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: rinse chromatographic column well back with the acetonitrile more than 50% and balance each other with 99%A phase+1%B and go up appearance, applied sample amount is 11.0g, and sample filtrating is diluted one times with pure water during last appearance.Linear gradient elution 45min collects the purpose peak, obtains purity greater than 80% above cut, the purpose peak cut of collecting is no more than in water temperature do second behind the intact acetonitrile of vacuum rotary steam under 35 ℃ the condition and go on foot purification of samples.
3. second go on foot the HPLC purifying:
Purification condition: chromatographic column: with the methyl-monosilane bonded silica gel is the chromatographic column of stationary phase, and 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 the balance appearance on the first step purifying cut with the acetonitrile more than 50%.Linear gradient elution 45min collects the purpose peak, obtains purity greater than 95% above cut, the purpose peak cut of collecting is no more than in water temperature do the 3rd behind 35 ℃ the intact acetonitrile of condition underspin and go on foot the desalting and purifying sample.
4. the 3rd go on foot the HPLC desalting and purifying: chromatographic column: with the methyl-monosilane bonded silica gel is the chromatographic column of stationary phase, and pillar diameter and length are: 100 mm * 250 mm.Concentration 0.2%, pH value are that the aqueous solution of 6.5 ammonium acetates is A 1 phase, and 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 in water temperature carries out lyophilize after vacuum rotary steam is concentrated into about 50 mg/ml under 35 ℃ the condition, can obtain purity and be 95% the smart peptide 7.1g of RAD-16II, the purifying total recovery is 64.5%.
Claims (6)
1. the purification process of a peptide biomaterial, said peptide biomaterial is RAD-16I or RAD-16II, this method may further comprise the steps:
Peptide biomaterial RAD-16I or RAD-16II adopt the trifluoracetic acid dissolving, cross and filter filtrating;
With cyanic acid silane group silica gel is the chromatographic column of stationary phase, and the filtrating of step 1) peptide biomaterial is gone up appearance with the pure water dilution; Adopting trifluoroacetic acid aqueous solution is the A phase, is that 0.05%-0.4% trifluoroacetic acetonitrile is the B phase with volumetric concentration, and linear gradient elution is collected purpose peak cut;
With the methyl-monosilane bonded silica gel is stationary phase, with step 2) appearance on the purification of samples 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;
With the methyl-monosilane bonded silica gel is the chromatographic column of stationary phase, appearance on the purification of samples that step 3) is obtained; With the volumetric concentration is 0.1%-0.2%, and 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 constant gradient wash-out 10-15min; And then be the A2 phase with the 0.1%-0.2% aqueous acetic acid, trifluoroacetic acid aqueous solution is the B phase, gradient is (1-10) %B+ (99-90) A2 constant gradient wash-out 10-15 minute, with sample wash-out lower prop, collects purpose peak cut; Dry smart peptide.
2. purification process as claimed in claim 1 is characterized in that: described step 2) step 1) peptide biomaterial is dissolved with pure trifluoracetic acid, the filtrating of gained is gone up appearance with the pure water dilution, and the multiple of dilution is 0.5-5 times.
3. purification process as claimed in claim 1 is characterized in that: the gradient of B phase is described step 2): 1%-30%.
4. like any described purification process of claim 1-3, it is characterized in that: the gradient of B phase is in the described step 3): 10%-40%.
5. like any described purification process of claim 1-3, it is characterized in that: with sample wash-out lower prop, the elutriant of employing is 50%A2+50%B in the described step 4).
6. purification process as claimed in claim 4 is characterized in that: with sample wash-out lower prop, the elutriant of employing is 50%A2+50%B in the described step 4).
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Citations (4)
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|---|---|---|---|---|
| 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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| Title |
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| ROBERTA GAMBARETTO等: "Self-Assembling Peptides: Sequence, Secondary Structure in Solution and Film Formation", 《BIOPOLYMERS》 * |
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