CN102675450A - Method for purifying thymosin beta4 - Google Patents
Method for purifying thymosin beta4 Download PDFInfo
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- CN102675450A CN102675450A CN201210146833XA CN201210146833A CN102675450A CN 102675450 A CN102675450 A CN 102675450A CN 201210146833X A CN201210146833X A CN 201210146833XA CN 201210146833 A CN201210146833 A CN 201210146833A CN 102675450 A CN102675450 A CN 102675450A
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- 238000000034 method Methods 0.000 title claims abstract description 29
- UGPMCIBIHRSCBV-XNBOLLIBSA-N Thymosin beta 4 Chemical compound N([C@@H](CC(O)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(O)=O)C(=O)[C@@H]1CCCN1C(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(C)=O UGPMCIBIHRSCBV-XNBOLLIBSA-N 0.000 title abstract description 11
- 102100035000 Thymosin beta-4 Human genes 0.000 title abstract description 7
- 108010079996 thymosin beta(4) Proteins 0.000 title abstract description 7
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 24
- 239000000741 silica gel Substances 0.000 claims abstract description 24
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 24
- 239000000243 solution Substances 0.000 claims abstract description 24
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 22
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims abstract description 9
- 239000008367 deionised water Substances 0.000 claims abstract description 9
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 9
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000012071 phase Substances 0.000 claims description 33
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
- 239000007864 aqueous solution Substances 0.000 claims description 16
- 150000003839 salts Chemical class 0.000 claims description 15
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 14
- 230000008859 change Effects 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 9
- 230000005526 G1 to G0 transition Effects 0.000 claims description 8
- 238000010828 elution Methods 0.000 claims description 8
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 6
- BLRPTPMANUNPDV-UHFFFAOYSA-N Silane Chemical group [SiH4] BLRPTPMANUNPDV-UHFFFAOYSA-N 0.000 claims description 4
- 238000010532 solid phase synthesis reaction Methods 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 2
- 238000001514 detection method Methods 0.000 claims description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 2
- IZGYIFFQBZWOLJ-CKAACLRMSA-N phaseic acid Chemical compound C1C(=O)C[C@@]2(C)OC[C@]1(C)[C@@]2(O)C=CC(/C)=C\C(O)=O IZGYIFFQBZWOLJ-CKAACLRMSA-N 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 239000002953 phosphate buffered saline Substances 0.000 claims description 2
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 abstract description 12
- 238000004007 reversed phase HPLC Methods 0.000 abstract description 2
- 238000010923 batch production Methods 0.000 abstract 1
- 238000001704 evaporation Methods 0.000 abstract 1
- 230000008020 evaporation Effects 0.000 abstract 1
- 238000004108 freeze drying Methods 0.000 abstract 1
- 239000008055 phosphate buffer solution Substances 0.000 abstract 1
- 102000004196 processed proteins & peptides Human genes 0.000 abstract 1
- 239000007787 solid Substances 0.000 abstract 1
- 238000009987 spinning Methods 0.000 abstract 1
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 abstract 1
- 230000002194 synthesizing effect Effects 0.000 abstract 1
- 230000001131 transforming effect Effects 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 12
- 238000000746 purification Methods 0.000 description 10
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 239000003814 drug Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 101000713575 Homo sapiens Tubulin beta-3 chain Proteins 0.000 description 3
- 108010078233 Thymalfasin Proteins 0.000 description 3
- 108010046075 Thymosin Proteins 0.000 description 3
- 102000007501 Thymosin Human genes 0.000 description 3
- 102100036790 Tubulin beta-3 chain Human genes 0.000 description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 description 3
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 description 3
- 238000004090 dissolution Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 238000005374 membrane filtration Methods 0.000 description 3
- 235000019837 monoammonium phosphate Nutrition 0.000 description 3
- 230000007935 neutral effect Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000010926 purge Methods 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 230000008719 thickening Effects 0.000 description 3
- NZVYCXVTEHPMHE-ZSUJOUNUSA-N thymalfasin Chemical compound CC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O NZVYCXVTEHPMHE-ZSUJOUNUSA-N 0.000 description 3
- 229960004231 thymalfasin Drugs 0.000 description 3
- LCJVIYPJPCBWKS-NXPQJCNCSA-N thymosin Chemical compound SC[C@@H](N)C(=O)N[C@H](CO)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CO)C(=O)N[C@H](CO)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H]([C@@H](C)CC)C(=O)N[C@H]([C@H](C)O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CC(O)=O)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCCCN)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](C(C)C)C(=O)N[C@H](C(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@H](CCC(O)=O)C(O)=O LCJVIYPJPCBWKS-NXPQJCNCSA-N 0.000 description 3
- 210000001541 thymus gland Anatomy 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 230000029663 wound healing Effects 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000035876 healing Effects 0.000 description 2
- 241000197194 Bulla Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 102400000800 Thymosin alpha-1 Human genes 0.000 description 1
- 208000025865 Ulcer Diseases 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000003453 ammonium sulfate precipitation method Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 208000002352 blister Diseases 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000003399 chemotactic effect Effects 0.000 description 1
- 229940126678 chinese medicines Drugs 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 201000010251 cutis laxa Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
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- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000009067 heart development Effects 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000037189 immune system physiology Effects 0.000 description 1
- 238000001155 isoelectric focusing Methods 0.000 description 1
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- 210000004165 myocardium Anatomy 0.000 description 1
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- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
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Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for purifying thymosin beta4 with a reversed phase high performance liquid chromatography, which is mainly used for solving the technical problem of low purity of thymosin beta4 obtained by separating in the prior art. According to the technical scheme of the invention, the method comprises the following steps of: (1) dissolving a crude peptide obtained by synthesizing solids with deionized water, gradually eluting and purifying by taking a reversed phase silica gel column of which the immobile phase is tetraalkylsilane bonded silica gel or octadecyl bonded silica gel and the mobile phase is a phosphate buffer solution as a phase A and taking chromatographically-pure acetonitrile as a phase B, and collecting a peptide solution of a target peak value; (2) switching and transforming into acetate by using an HPLC (High Performance Liquid Chromatography) method; and (3) and performing spinning evaporation, concentration and freeze drying on a final high-purity peptide solution under reduced pressure to obtain a powdery finished peptide. The invention provides a method for purifying thymosin beta4, which is suitable for industrialization. According to the method, fine peptides of which the purities are over 98.0 percent can be obtained, batch production is available, and the requirements of high purity, high yield, low cost and high efficiency are met.
Description
Technical field
The invention belongs to the reversed-phase HPLC technical field, especially relate to the method for a kind of mass industrialization purifying extrasin beta 4 (Thymosin β 4).
Background technology
Extrasin beta 4 is from thymus gland, to separate the eighties in 20th century to obtain, find afterwards all extensive a kind of small peptide material that exists in many tissues; It is a kind of important ABP; Contain 43 amino acid; The about 5kD of molecular weight, iso-electric point is 5.1, in Mammals, has higher conservative property.Extrasin beta 4 is a kind of small molecular protein of being made up of 43 amino acid, and having very strong epithelial cell stimulates and regeneration, to playing an important role in the brephic heart development.(thymosin-β 4 is from calf thymus component V, to separate to purify to obtain Tb4) to Zadaxin-β 4 at first.Find behind the extrasin beta 4 to be present in the nearly all somatocyte except red corpuscle, platelet content is the highest, and distribution is also arranged in blood plasma and tissue juice, has stronger epithelium repair.At present, U.S. Regene RX company exploitation chemosynthesis extrasin beta-4, the research of its " bulla property cutis laxa " has got into the II clinical trial phase.
In recent years, the promotion wound healing effect of extrasin beta 4 receives publicity.Nearest discovers, in the wound healing process of cardiac muscle, cornea and skin, extrasin beta 4 mainly is as a kind of chemically induced factor, regulates the expression of multiple growth factor and the migration of chemotactic neonatal cell.Because the ability of body self healing is extremely limited; In case taking place, damage or pathology be difficult to self-healing; Even cause ulcer or cicatrization; Need the reparation of outer rim property more or start the endogenous reparation, but do not find effectively to promote the medicine of wound healing as yet at present, extrasin beta 4 is that the research of medicine for healing wound provides a new direction.
Initial from calf thymus, separate the thymosin 5 that obtains (Thymosin fration 5, TF5), according to its again isoelectric focusing electrophoresis analyze the position on the collection of illustrative plates, can be divided into three districts of α β γ: α district pI 5, β district pI <between 5 ~ 7, γ district pI>7.Can the enhancing body cellular immune function like Thymosin alpha 1, be used for clinical treatment.Current research finds that extrasin beta 4 (Thymosin β 4, T β 4) is not only participated in immunologic process, and shows multi-biological function, has caused investigator's extensive concern.Just because of the biological applications prospect of T β 4 these a series of Chinese medicines of product, separation and purification obtains T β 4 products and is used for further scientific research and just seems particularly important.In China, less relatively at present for the research of T β 4 separation purifying techniques." Chinese Journal of New Drugs " 2008 the 22nd phases: adopt ammonium sulfate precipitation method to make thymosin 5 and slightly carry article, utilize zwitterion exchange chromatography purifying to obtain Zadaxin β 4 products then, and adopt SDS-PAGE and HPLC method to carry out Analysis and Identification.The result: it is 90.1% product that separation and purification has obtained purity.The be nowhere near purity requirement of present drug research of the purity of the Zadaxin β 4 of this method gained.
At present; The domestic reversed-phased high performace liquid chromatographic of utilizing is quite few to the document that the extrasin beta 4 of solid phase synthesis carries out separation and purification; So utilize reversed-phased high performace liquid chromatographic the extrasin beta 4 of solid phase synthesis is carried out separation and purification; Obtain a large amount of highly purified extrasin beta 4s, become to be badly in need of the technical problem of solution.The present invention has not only solved at present from body tissue to separate and has obtained the difficulty of extrasin beta 4, and can obtain meeting the required high purity finished product of drug research, to its in the research of medicine and using of it can extend efficient help.
Summary of the invention
The object of the present invention is to provide a method that is suitable for the pure extrasin beta 4 of industrialization, mainly solve the prior art separation and obtain the lower technical problem of extrasin beta 4 purity.
For realizing above-mentioned purpose, technical scheme of the present invention is following: a kind of method of purifying extrasin beta 4 comprises the steps:
1) the thick peptide of solid phase synthesis gained is used deionized water dissolving, stationary phase is the reverse phase silica gel post, and moving phase is that phosphate buffer soln is the A phase, and trifluoroacetic acid aqueous solution is the B phase, carries out the gradient elution purifying, collects the peptide solution of purpose peak value;
2) adopt HPLC to change the exchange of salt method the high purity peptide solution behind the purifying and change into acetate;
3) final high purity peptide solution vacuum rotary steam concentrated frozen is dry, get Powdered finished product.
More preferred scheme is: the pH of the phosphate buffered saline buffer of said A phase is 3.5 ~ 4.8.
More preferred scheme is: the mass percentage concentration of said trifluoroacetic acid aqueous solution is 20-65%, and the detection wavelength is 220nm.
More preferred scheme is: described reverse phase silica gel post is the reverse phase silica gel of tetraalkyl silane group silica gel or octadecyl silane.
More preferred scheme is: described HPLC changes the salt method, and pH is 2.0 ~ 3.0 the acetum and the two phase liquid of chromatographically pure methyl alcohol, and chromatographic column is the reverse phase silica gel post of tetraalkyl silane group silica gel or octadecyl silane.
The invention has the beneficial effects as follows: a process method that is suitable for the pure extrasin beta 4 of industrialization of proposition of the present invention, use reversed-phased high performace liquid chromatographic purifying extrasin beta 4, and change the exchange of salt method with HPLC and change salt; Can not only obtain HPLC purity greater than 98.0% smart peptide; And can mass production, reaching the purity height, yield is high; Cost is low, the requirement that efficient is fast.
Embodiment
Embodiment 1
1. sample preparation: the extrasin beta 4 that will synthesize gained is with deionized water dissolving (concentration of ordinary dissolution is about 50mg/ml), collects filtrate for later use after using the aperture as the 0.45um membrane filtration.
2. purifying
Purification condition: chromatographic column: with the silica gel bonded silica gel of tetraalkyl is the chromatographic column of stationary phase, and the pillar diameter with length is: 5cm * 25cm. moving phase: A mutually: ammonium dihydrogen phosphate aqueous solution is transferred pH to 3.0 ~ 4.8 with analytically pure phosphoric acid solution; B phase: trifluoroacetic acid aqueous solution.Flow velocity: 40-55ml/min.Detect wavelength: 220nm.Gradient: B%:20-60%, 30-50min.Sample size is 1.1-1.8g;
Purge process: rinse chromatographic column well back with the acetonitrile of mass percentage concentration more than 60% and go up appearance, applied sample amount is a sample solution.Linear gradient elution is collected the purpose peak, places in the receiving flask subsequent use with collecting good peptide solution.
3. commentaries on classics salt: use the reverse phase silica gel post of the deionized water rinsing stationary phase of mass percentage concentration more than 60% as the tetraalkyl bonded silica gel; Rinsing back (basically neutral) well, to use the pH that configures again be 2.0 ~ 3.5 acetate aqueous solutions and chromatographically pure methyl alcohol two phase liquid balance chromatographic column; Treat that beginning to go up appearance after the chromatographic column balance thoroughly changes salt; The purpose peptide solution of applied sample amount for having collected; Linear gradient elution: with the acetate aqueous solution of 10-20min mass percentage concentration 60-80%, through the methanol solution of 30-60min mass percentage concentration 15-75%, collect whole target peaks more earlier.
4. will change all peptide solutions of gained after the salt, and be evaporated to 1g/50ml, thickening temperature is no more than 37 ℃, and lyophilize gets purity greater than 98.0% extrasin beta 4 then, and purification yield can get more than 48%.
Embodiment 2
1. sample preparation: the extrasin beta 4 that will synthesize gained is with deionized water dissolving (concentration of ordinary dissolution is about 50mg/ml), collects filtrate for later use after using the aperture as the 0.45um membrane filtration.
2. purifying:
Purification condition: chromatographic column: with the 18 alkyl silica gel bonded silica gel is the chromatographic column of stationary phase, and the pillar diameter with length is: 5cm * 25cm. moving phase: A mutually: ammonium dihydrogen phosphate aqueous solution is transferred pH to 3.5 ~ 5.3 with analytically pure phosphoric acid solution; B phase: trifluoroacetic acid aqueous solution.Flow velocity: 40-55ml/min.Detect wavelength: 220nm.Gradient: B%:23-65%, 40-60min.Sample size is 1.0-1.7g;
Purge process: rinse chromatographic column well back with the acetonitrile of mass percentage concentration more than 60% and go up appearance, applied sample amount is a sample solution.Linear gradient elution is collected the purpose peak, places in the receiving flask subsequent use with collecting good peptide solution.
3. commentaries on classics salt: use the reverse phase silica gel post of the deionized water rinsing stationary phase of mass percentage concentration more than 60% as octadecyl silane; Rinsing back (basically neutral) well, to use the pH that configures again be 2.5 ~ 3.5 acetate aqueous solutions and chromatographically pure methyl alcohol two phase liquid balance chromatographic column; Treat that beginning to go up appearance after the chromatographic column balance thoroughly changes salt; The purpose peptide solution of applied sample amount for having collected; Linear gradient elution: with the acetate aqueous solution of 10-30min mass percentage concentration 60-80%, through the methanol solution of 40-70min mass percentage concentration 20-80%, collect whole target peaks more earlier.
4. will change all peptide solutions of gained after the salt, and be evaporated to 1g/50ml, thickening temperature is no more than 37 ℃, and lyophilize gets purity greater than 98.0% extrasin beta 4 then, and purification yield can get more than 55%.
Embodiment 3
1. sample preparation: the extrasin beta 4 that will synthesize gained is with deionized water dissolving (concentration of ordinary dissolution is about 50mg/ml), collects filtrate for later use after using the aperture as the 0.45um membrane filtration.
2. purifying:
Purification condition: chromatographic column: with the 18 alkyl silica gel bonded silica gel is the chromatographic column of stationary phase, and the pillar diameter with length is: 10cm * 50cm. moving phase: A mutually: ammonium dihydrogen phosphate aqueous solution is transferred pH to 3.5 ~ 4.8 with analytically pure phosphoric acid solution; B phase: trifluoroacetic acid aqueous solution.Flow velocity: 60-80ml/min.Detect wavelength: 220nm.Gradient: B%:20-65%, 60-90min.Sample size is 8.0-10.0g;
Purge process: rinse chromatographic column well back with the acetonitrile more than 60% and go up appearance, applied sample amount is a sample solution.Linear gradient elution is collected the purpose peak, places in the receiving flask subsequent use with collecting good peptide solution.
3. commentaries on classics salt: use the reverse phase silica gel post of the deionized water rinsing stationary phase of mass percentage concentration more than 60% as octadecyl silane; Rinsing back (basically neutral) well, to use the pH that configures again be 2.0 ~ 3.0 acetate aqueous solutions and chromatographically pure methyl alcohol two phase liquid balance chromatographic column; Treat that beginning to go up appearance after the chromatographic column balance thoroughly changes salt; The purpose peptide solution of applied sample amount for having collected; Linear gradient elution: with the acetate aqueous solution of 10-30min mass percentage concentration 60-80%, through the methanol solution of 50-100min mass percentage concentration 20-80%, collect whole target peaks more earlier.
4. will change all peptide solutions of gained after the salt, and be evaporated to 1g/50ml, thickening temperature is no more than 37 ℃, and lyophilize gets purity greater than 98.0% extrasin beta 4 then, and purification yield can get more than 60%.
Claims (5)
1. the method for a purifying extrasin beta 4 adopts reversed-phased high performace liquid chromatographic, it is characterized in that comprising the steps:
1) the thick peptide of solid phase synthesis gained is used deionized water dissolving, stationary phase is the reverse phase silica gel post, and moving phase has two phases, and phosphate buffer soln is the A phase, and trifluoroacetic acid aqueous solution is the B phase, carries out the gradient elution purifying, collects the peptide solution of purpose peak value;
2) adopt HPLC to change the exchange of salt method the high purity peptide solution behind the purifying and change into acetate;
3) final high purity peptide solution vacuum rotary steam concentrated frozen is dry, get Powdered finished product.
2. according to the method for the said purifying extrasin beta 4 of claim 1, it is characterized in that: the pH of said A phase phosphate buffered saline buffer is 3.5 ~ 4.8.
3. according to the method for the said purifying extrasin beta 4 of claim 1, it is characterized in that: the mass percentage concentration of said B phase trifluoroacetic acid aqueous solution is 20-65%, and the detection wavelength is 220nm.
4. according to the method for the said purifying extrasin beta 4 of claim 1, it is characterized in that: described reverse phase silica gel post is tetraalkyl silane group silica gel or octadecyl silane.
5. according to the method for the said purifying extrasin beta 4 of claim 1; It is characterized in that: described HPLC changes the salt method; PH is 2.0 ~ 3.0 the acetum and the two phase liquid of chromatographically pure methyl alcohol, and chromatographic column is the reverse phase silica gel post of tetraalkyl silane group silica gel or octadecyl silane.
Priority Applications (1)
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102827258A (en) * | 2012-10-08 | 2012-12-19 | 吉尔生化（上海）有限公司 | Method for purifying Enfuvirtide |
| CN104829695A (en) * | 2015-05-04 | 2015-08-12 | 吉尔生化（上海）有限公司 | Purifying method for alarelin |
| CN105891399A (en) * | 2015-04-03 | 2016-08-24 | 北京诺思兰德生物技术股份有限公司 | Method for detecting thymosin beta 4 content based on high performance liquid chromatograph |
| CN112851746A (en) * | 2019-11-26 | 2021-05-28 | 深圳翰宇药业股份有限公司 | Desalination method utilizing freeze-drying principle |
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| CN101798335A (en) * | 2010-03-30 | 2010-08-11 | 深圳翰宇药业股份有限公司 | Purification method of thymosin extrasin alpha 1 |
| CN102167738A (en) * | 2011-01-26 | 2011-08-31 | 浙江大学 | Method for separating and purifying thymosin beta4 |
| CN102382188A (en) * | 2011-11-07 | 2012-03-21 | 深圳翰宇药业股份有限公司 | Method for preparing carperitide acetate |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101798335A (en) * | 2010-03-30 | 2010-08-11 | 深圳翰宇药业股份有限公司 | Purification method of thymosin extrasin alpha 1 |
| CN102167738A (en) * | 2011-01-26 | 2011-08-31 | 浙江大学 | Method for separating and purifying thymosin beta4 |
| CN102382188A (en) * | 2011-11-07 | 2012-03-21 | 深圳翰宇药业股份有限公司 | Method for preparing carperitide acetate |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102827258A (en) * | 2012-10-08 | 2012-12-19 | 吉尔生化（上海）有限公司 | Method for purifying Enfuvirtide |
| CN105891399A (en) * | 2015-04-03 | 2016-08-24 | 北京诺思兰德生物技术股份有限公司 | Method for detecting thymosin beta 4 content based on high performance liquid chromatograph |
| CN104829695A (en) * | 2015-05-04 | 2015-08-12 | 吉尔生化（上海）有限公司 | Purifying method for alarelin |
| CN104829695B (en) * | 2015-05-04 | 2018-09-07 | 吉尔生化（上海）有限公司 | A method of purifying alarelin |
| CN112851746A (en) * | 2019-11-26 | 2021-05-28 | 深圳翰宇药业股份有限公司 | Desalination method utilizing freeze-drying principle |
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