CN102477094A - Separation and purification process for synthetic thymosin alpha 1 - Google Patents
Separation and purification process for synthetic thymosin alpha 1 Download PDFInfo
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Abstract
The invention relates to a purification process for thymosin alpha 1 after chemical synthesis. Specifically speaking, the conventional manner of combined ion exchange and vacuum condensation for desalination and condensation of thymosin alpha 1 obtained through chemical synthesis is changed into one-shot nanofiltration in the invention. The process provided in the invention has the advantages of simpleness, high efficiency, low energy consumption and suitability for industrial production.
Description
Technical field
The present invention relates to a kind of separation purification method of synthetic Thymosin alpha 1, belong to biologics preparation technology field.
Background technology
Thymosin alpha 1 (T α 1) is the main bioactive ingredients of thymosin; Be immune regulator important in the human body; Research shows; Thymosin alpha 1 promotes the growth of marrow liver cell to be divided into lymphoblast and prolymphocyte, and the inducer T lymphocyte differentiation makes mature T cell further be divided into lymphoblast and prolymphocyte with ripe; The inducer T lymphocyte differentiation makes mature T cell further be divided into several different subgroups with ripe, like killer cell, memory cell, effector cell, helper T lymphocyte etc., and produces various solubility media; Strengthen lymphocyte to mitotic reaction, and can increase the synthetic of adenoid protein and nucleic acid; Increase the generation of IFN-γ, IFN-α, IL-2, IL-3 and lymphotoxin, strengthen antiviral and antineoplastic immunoreation; Suppression of autoimmune responses; Recover the function of suppressor T cell.Thymosin alpha 1 has been widely used in clinical, treats multiple disease, as be used for chronic hepatitis B treatment, be used for the treatment of hepatitis C, hepatocellular carcinoma, malignant melanoma; Be used to treat the treatment of infection, autoimmune disorder, malignant tumour etc.
Zadaxin is a kind of biologically active polypeptides of being made up of 28 amino acid, and iso-electric point is 4.2, and molecular weight is 3108 dalton.At present, both at home and abroad the working method of T α 1 mainly contains 3 kinds, i.e. tissue extraction method, gene engineering research and chemical synthesis process, and wherein, the solid state chemistry synthesis method prepares T α 1 has become one of mainstream technology.Cheng Hu (Cheng Hu, Wei Ping, Zhu Yishen. the solid phase synthesis thymosin. Nanjing University of Technology's journal [J] .2004,26 (2): it is initial 78) utilizing Fmoc-Asn (Trt)-Wang resin, the synthetic Thymosin alpha 1 that obtains.Han Xiang (Han Xiang, Gu Jun, Yuan Qinglan, etc. the solid phase synthesis of human thymosin alfa 1 and external activity research. Chinese pharmaceutical chemistry magazine [J] .2004,14 (1): 27) also through adopting the synthetic human thymosin alfa 1 that obtains of Fmoc-Asn (Trt)-Wang resin solid-phase resin.
Because in the middle of synthetic T α 1 process of Fmoc solid phase method; The joint efficiency in each step can not reach 100%; Problems such as various side reactions, racemization appear in middle meeting; Make that synthetic this polypeptide is a kind of thick product of low-purity, these impurity comprise: the formed by product of incomplete deprotection of diastereoisomeric (racemize) polypeptide, disappearance (not exclusively) peptide, fracture peptide, byproduct of reaction, oxidation peptide, amino acid side chain, used toxic agent and solvent etc. in synthesizing, these by products have with the title product structure on very close material; Textural difference is little; Physicochemical property are also very approaching, just increased the difficulty and the cost of separation and purification like this, as occurring racemization impurity easily at 28 Ser; Its physico-chemical property and target T α 1 and similar, so the separation and purification problem also is a biggest obstacle in the chemically synthesized polypeptide.
At present, the conventional separation method of chemically synthesized polypeptide is the separation method that adopts compounds such as macromole such as protein, and its conventional means has three kinds: gel filtration chromatography, ion-exchange chromatography, RPLC.The purification process that separates solid phase synthesis T α 1 adopts two-step separation more, promptly at first adopts ion-exchange chromatography to remove the overwhelming majority's in the bullion impurity, then; Adopt RPLC to carry out last purifying again, again the T α behind the purifying in the performance liquid 1 is carried out vacuum decompression and concentrate, remove organic solvent wherein; As the acetonitrile that often can use, and because in commercial process, the medicine liquid volume of producing preparation is big; Adopt the rotation vacuum concentration, need carry out at a certain temperature, but peptide or protein medicaments should not concentrate under higher temperature again; Can only control low relatively temperature and carry out vacuum concentration; It is long to concentrate the required time, and peptide or protein medicaments be under the situation of long-time heating, easy sex change inactivation.
Although nanofiltration is used very wide, it is when separation or concentrated oligopeptides; All be to concentrate through nanofiltration to remove some small molecules amino acid or available nf membrane fractionated small peptide; Perhaps to the holding back of monovalent salt or a polyvalent salt, and, all be usually with the vacuum concentration technology to being mixed in the organic solvent in polypeptide or the albumen; Remove organic solvent wherein, do not see the method application of adopting nanofiltration to remove the organic solvent in the peptide.Chinese patent CN1152052C also discloses a kind of process method for preparing Zadaxin; It comprises to fresh thymus gland homogenate carry out fragmentation, centrifugal, ultrafiltration molecular weight cut-off is 6000-10000; And then it is concentrated that ultrafiltrated is carried out nanofiltration, removes the water soluble component of some inorganic salt, small peptide and amino acids.
Summary of the invention
The object of the present invention is to provide the method for a kind of separation and purification synthetic T α 1 (Thymosin alpha 1); Among the present invention, when concentrating, adopt the mode of membrane-concentrated through consummate T α 1 solution of reversed phase high efficiency liquid phase; Be specially and adopt the spissated mode of nf membrane to concentrate to remove small molecular weight impurity; As in liquid phases separation, bringing next organic solvent acetonitrile into, thickening efficiency is high, and the time is short; And only need at normal temperature even be lower than in the environment of normal temperature just can carry out, greatly reduce T α 1 long-term contact and cause the risk of T α 1 sex change inefficacy under hot environment.
Particularly, the present invention relates to:
1, a kind of separation purification method of synthetic Thymosin alpha 1 comprises
(1) with the thick peptide of ion-exchange chromatogram purification synthetic;
(2) polypeptide of the further refining purifying of reversed-phase liquid chromatography after ion-exchange chromatography is handled;
(3) polypeptide after will making with extra care concentrates, and removes organic solvent wherein;
(4) liquid concentrator changes salt, lyophilization gets finished product;
Wherein, described concentrating is to adopt membrane filtration to concentrate.
2, it is that nf membrane concentrates that the separation purification method in the item 1, wherein said membrane filtration concentrate, and the nf membrane aperture is 50-1000 dalton.
3, the separation purification method in the item 1, in the thick peptide of wherein said IX column purification synthetic, used ion exchange column is an anion-exchange column.
4,3 a described separation purification method, wherein said anion-exchange column is the weak anionic exchange column.
5,4 a described separation purification method, wherein said weak anionic exchange column is a DEAE sepharoseFast Flow ion exchange column.
6,1 described separation purification method, wherein the elutriant of anion-exchange column is that pH is that 5.8 PB buffer A and pH are 5.8 PBS buffer B, gradient elution.
7, the separation purification method under the item 1, wherein the reversed-phase liquid chromatography post is a C18 reversed-phase liquid chromatography post.
8,1 described separation purification method, wherein the reversed-phase liquid chromatography elution system is 12% the acetonitrile solution that contains 0.1%TFA and 20% the acetonitrile solution system that contains 0.1%TFA, gradient elution.
9,1 a described separation purification method, for adopting reverse-phase chromatography to change salt, the commentaries on classics salt buffer is an acetum in the wherein said commentaries on classics salt step, the commentaries on classics salt eluent is an acetonitrile solution.
10,9 a described separation purification method, wherein said acetate buffer solution is the acetum of 0.05-0.5mol/L, described acetonitrile solution is the acetonitrile solution of mass percent 5%-30%.
In item 1 or item 2, concentrating the film that adopts is the filter membrane of organic solvent-resistant.
Embodiment
The objective of the invention is to be to provide a kind of preparation technology that can improve the Thymosin alpha 1 industrial production efficiency; The technology that the present invention adopted is that the thick peptide (T α 1 thick peptide) through solid phase synthesis is removed more most impurity through ion exchange column chromatography earlier; T α 1 thick peptide behind the chromatography of ions purifying; With the refining purifying of reversed-phase liquid chromatography post, T α 1 solution after the reversed-phase liquid chromatography post is refining adopts the mode of membrane filtration to concentrate to remove organic solvent again; To accelerate thickening efficiency, reduce the risk of the T α 1 long-time sex change that under hot environment, causes.
Among the present invention, term " membrane filtration concentrate " is that a kind of employing ultra-filtration membrane carries out molecular retention, with different sized molecules through different filter membrane apertures to reach concentrated and purified purpose.When concentrating with membrane filtration, can be under condition of normal pressure, to carry out filtering and concentrating, also can be filtering and concentrating under the condition of pressurization.In the present invention; The membrane filtration mode that is adopted is that nf membrane is filtered, and term " nf membrane " is meant a kind of semi-permeable membranes, a kind of functional semi-permeable membranes that can allow solvent molecule or some low molecular weight solutes or low price ion to see through; T α 1 after this is used for concentrating purifying of the present invention; The filter membrane pore size is 50-1000 dalton, is preferably 50-500 dalton, more preferably 50-200 dalton.Used film is the film of organic solvent-resistant.
Among the present invention, term " ion exchange resin " can be to use Zeo-karb, such as 732 type Zeo-karbs, D001 large hole cation exchanger resin; Also can be anionite-exchange resin; Like 330 alkaline epoxy type anion exchange resins, D301 macropore basic anion exchange resin, DEAE sepharose FastFlow anionite-exchange resin; Among the present invention, be preferably anionite-exchange resin, more preferably weak anion exchange resin.
To the thick peptide of solid phase method synthetic, when carrying out preliminary purification with anionite-exchange resin; Ion exchange elution circuit; Can be to mix elution system with commonly used some in this area to carry out gradient elution, as being the mixing system gradient elution of buffered soln such as PB-PBS elution system, Tris-HCl and Tris-HCl-NaCl elution system.PB is meant phosphate buffered saline buffer; It is the composite by a certain percentage phosphate buffered saline buffer of SODIUM PHOSPHATE, MONOBASIC and Sodium phosphate, dibasic; The ratio of specific configuration can be adjusted according to the difference of the pH value of required configuration, and the collocation method of sort buffer solution is well-known to those skilled in the art; PBS buffered soln is in PB buffered soln, to add a certain amount of NaCl, can obtain.
Among the present invention; The reversed-phase liquid chromatography post of being selected for use is preferably C18 reversed phase high efficiency liquid phase preparative hplc post; T α behind ion-exchange purification 1 is carried out further refining purifying; Removing the wherein extremely similar assorted peptide of structure, character, the reversed-phase liquid chromatography flow phase system is 12% the acetonitrile solution that contains 0.1%TFA and 20% the acetonitrile solution system that contains 0.1%TFA, gradient elution.
Among the present invention, further comprise the step of changeing salt, to remove the harmful acid ion of in refining purge process, bringing into, like the TFA acid ion.Described commentaries on classics salt employing reverse-phase chromatography changes the salt method, is about to reversed-phase column on T α 1 solution after nanofiltration concentrates, with configured in advance good commentaries on classics salt buffer and commentaries on classics salt eluent wash-out; Collect the target peak part, promptly get T α 1 solution, T α 1 solution of this moment has been removed the TFA acid ion; Volume is also less relatively; Concentrate once more when removing organic solvent (like acetonitrile), concentrating means can be to adopt the vacuum decompression method of enrichment, also can be to concentrate with nf membrane.
The thick T α 1 of embodiment 1 ion-exchange chromatogram purification solid phase synthesis
Purifying is prepared: prepare vessel, 250 ℃ of xeothermic depyrogenations or water for injection clean; Preparation column balance buffering liquid A (20mmol/L PB, pH 5.8) and post elution buffer B (20mmol/L PB, 500mmol/L sodium-chlor, pH5.8), for use; It is an amount of to get T α 1 thick peptide, adds column balance buffering liquid and is dissolved to about 10mg/ml, and 0.45 μ m filters and processes thick peptide liquid, and is for use; Prepare AKTABASIC10 preparative liquid chromatography system, medium is DEAEsepharose Fast Flow (anionite-exchange resin, a preferred weak anion exchange resin).The liquid-inlet of A pump is put into column balance buffering liquid, the liquid-inlet of B pump is put into the post elution buffer.Power up, open driving, with column balance buffering liquid balance chromatographic column, UV-detector is set 220nm, transfers flow velocity 100ml/min, and is to the baseline balance, for use.
Target peak is collected: calculate according to appearance 20mg amount on every mL media, with appearance on the thick peptide solution.Carry out wash-out according to the gradient in the following table.When the peak height at first peak that step 3 wash-out goes out begins to collect during greater than 100mAU, when being reduced to 100mAU under the peak height, stop to collect.Continue wash-out, accomplish until elution step.Collect part and be T α 1 collection liquid I.
| Step | A(％) | B(％) | Volume (column volume) |
| 1 | 100 | 0 | 2 |
| 2 | 90 | 10 | 6 |
| 3 | 90→60 | 10→40 | 5 |
| 4 | 60→0 | 40→100 | 1 |
| 5 | 0 | 100 | 1 |
Embodiment 2 reverse-phase chromatographies are consummate
Prepare: prepare vessel, 250 ℃ of xeothermic depyrogenations or water for injection clean; Preparation mobile phase A (12% acetonitrile solution, contain 0.1%TFA) and Mobile phase B (25% acetonitrile solution, contain 0.1%TFA), 0.45 μ m filtration, for use; Get T α 1 and collect liquid I, add acetonitrile, the final concentration that makes acetonitrile is 12%, and TFA content is that peptide liquid is processed in 0.1%, 0.45 μ m filtration, and is for use; Prepare Tianjin, island LC-20A preparative liquid chromatography system, filler C18 powers up, and opens driving, advances mobile phase A, and UV-detector is set 220nm, transfers flow velocity 50ml/min, and is to the baseline balance, for use.
C18 reverse-phase chromatography purifying is collected: get the T α 1 that handles well and collect liquid I liquid 100ml sample introduction, flow velocity is constant, walks the chromatogram program according to the gradient in the following table.(10 μ mC18) observes absorption value and changes, and removes assorted peak, the appearance of close observation main peak during about 25min; Collect the main peak elutriant when beginning to rise to about 100mv immediately, descending stops when being less than about 100mv collecting, and proceeds to 45min and finishes; The circulation inserting needle is collected with method.Collect liquid and detect qualified (purity＞97%) with the analysis mode high performance liquid chromatograph; Then follow-up with method collection main peak, make purity qualified otherwise should adjust collection main peak collection liquid middle portion, after treating all to separate completion; Merge each time main peak and collect liquid, promptly T α 1 collects liquid II.
| Time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 100 | 0 |
| 20 | 80 | 20 |
| 35 | 68 | 32 |
| 40 | 0 | 100 |
| 50 | 0 | 100 |
Concentrating of embodiment 3 refining T α 1 solution
1 rotation vacuum concentration
Get the consummate T α 1 solution 20L of RPLC of embodiment gained, at about 40 ℃ of rotation vacuum concentration, the rotation concentration tank is 10L, and T α 1 consummate liquid is concentrated into 1/3 of about original volume, needs consuming time more than at least 10 hours.
2 nanofiltrations concentrate
Prepare collecting and filtering apparatus, nf membrane aperture 100 dalton; Flow rate pump is regulated in start, keeps film intake pressure 1.2Mpa, and film top hole pressure 1.0Mpa makes feed liquid be concentrated into 1/3 of original volume, adds purified water to original volume.Continue to be concentrated into 1/3 of original volume, concentrate to finish, concentrate about 30 minutes of required time, significantly reduce than time of rotation vacuum concentration, be fit to very much preparation of industrialization production.
Embodiment 4 changes salt
Prepare: prepare vessel, 250 ℃ of xeothermic depyrogenations or water for injection clean; Preparation is changeed salt buffer (0.1mol/L acetum) and is changeed salt eluent (20% acetonitrile solution), and 0.45 μ m filters, and is for use.Get T α 1 refined solution, add glacial acetic acid, make the final concentration of acetic acid reach 5%, 0.45 μ m filtration, for use.Prepare Tianjin, island LC-20A preparative liquid chromatography system, filler C18 powers up, and opens driving, advances to change salt buffer, and UV-detector is set 220nm, transfers flow velocity 50ml/min, and is to the baseline balance, for use.
Change salt: get the T α 1 refined solution 100ml sample introduction of handling well.After accomplishing, sample introduction, washes 2 column volumes with changeing salt buffer flushing post bed.With water for injection flushing post bed, flush volume is 2 column volumes then.With changeing the salt eluent wash-out, pay close attention to the appearance of main peak then, when main peak rises to about 100mv, collect the main peak elutriant immediately, descending stops when being less than about 100mv collecting.With the method multi-pass operations, collect main peak and merging.Promptly get T α 1 and collect liquid III.
Concentrate: get above-mentioned T α 1 and collect liquid III, 37 ℃ are rotated about 10 times of vacuum concentration, and promptly T α 1 refined liquid detects, and is subsequent use.
Freeze-drying: T α 1 refined liquid 0.2 μ m is filtered, filtrating packing freeze-drying dish, liquid thickness is no more than 1.2cm, advances freeze drier and carries out freeze-drying by the freeze-drying program of setting, and promptly gets.
Claims (10)
1. the separation purification method of a synthetic Thymosin alpha 1 comprises
(1) with the thick peptide of ion-exchange chromatogram purification synthetic;
(2) polypeptide of the further refining purifying of reversed-phase liquid chromatography after ion-exchange chromatography is handled;
(3) polypeptide after will making with extra care concentrates, and removes organic solvent wherein;
(4) liquid concentrator changes salt, lyophilization gets finished product;
Wherein, described concentrating is to adopt membrane filtration to concentrate.
2. it is that nf membrane concentrates that the separation purification method in the claim 1, wherein said membrane filtration concentrate, and the nf membrane aperture is 50-1000 dalton.
3. claim 1 or 2 described separation purification method, the used film of wherein said membrane filtration is the filter membrane of organic solvent-resistant.
4. the described separation purification method of claim 1, in the thick peptide of wherein said IX column purification synthetic, used ion exchange column is an anion-exchange column.
5. the described separation purification method of claim 3, wherein said anion-exchange column is a DEAEsepharose Fast Flow ion exchange column.
6. the described separation purification method of claim 1, wherein the elutriant of anion-exchange column is that pH is that 5.8 PB buffer A and pH are 5.8 PBS buffer B, gradient elution.
7. the separation purification method under the claim 1, wherein the reversed-phase liquid chromatography post is a C18 reversed-phase liquid chromatography post.
8. the described separation purification method of claim 1, wherein the reversed-phase liquid chromatography elution system is 12% the acetonitrile solution that contains 0.1%TFA and 20% the acetonitrile solution system that contains 0.1%TFA, gradient elution.
9. the described separation purification method of claim 1 is employing reverse-phase chromatography commentaries on classics salt in the wherein said commentaries on classics salt step, and the commentaries on classics salt buffer is an acetum, and the commentaries on classics salt eluent is an acetonitrile solution.
10. the described separation purification method of claim 9, wherein said acetate buffer solution is the acetum of 0.05-0.5mol/L, described acetonitrile solution is the acetonitrile solution of mass percent 5%-30%.
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Cited By (6)
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| CN103048395A (en) * | 2012-07-12 | 2013-04-17 | 海南合瑞制药股份有限公司 | Deletion peptide of 20 peptide phase for solid-phase synthesis of thymosin alpha 1, non-peptide impurity and detection method |
| CN103163236A (en) * | 2012-07-12 | 2013-06-19 | 海南合瑞制药股份有限公司 | Method for identifying whether purified thymosin alpha 1 contains deletion peptide or not |
| CN103163233A (en) * | 2012-07-12 | 2013-06-19 | 海南合瑞制药股份有限公司 | Deletion peptide and detection method for peptide phase 28 of solid-phase synthesis thymulin alpha1 |
| CN103163235A (en) * | 2012-07-12 | 2013-06-19 | 海南合瑞制药股份有限公司 | Deletion peptide and detection method for peptide phase 16 of solid-phase synthesis thymulin alpha1 |
| CN109705206A (en) * | 2019-03-12 | 2019-05-03 | 江苏诺泰澳赛诺生物制药股份有限公司 | A kind of purification process and purposes of thymalfasin |
| WO2020077927A1 (en) * | 2018-10-14 | 2020-04-23 | 深圳市健元医药科技有限公司 | Post-processing method for gonadotropin-releasing hormone antagonist |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103048395A (en) * | 2012-07-12 | 2013-04-17 | 海南合瑞制药股份有限公司 | Deletion peptide of 20 peptide phase for solid-phase synthesis of thymosin alpha 1, non-peptide impurity and detection method |
| CN103163236A (en) * | 2012-07-12 | 2013-06-19 | 海南合瑞制药股份有限公司 | Method for identifying whether purified thymosin alpha 1 contains deletion peptide or not |
| CN103163233A (en) * | 2012-07-12 | 2013-06-19 | 海南合瑞制药股份有限公司 | Deletion peptide and detection method for peptide phase 28 of solid-phase synthesis thymulin alpha1 |
| CN103163235A (en) * | 2012-07-12 | 2013-06-19 | 海南合瑞制药股份有限公司 | Deletion peptide and detection method for peptide phase 16 of solid-phase synthesis thymulin alpha1 |
| CN103048395B (en) * | 2012-07-12 | 2014-06-04 | 海南合瑞制药股份有限公司 | Deletion peptide of 20 peptide phase for solid-phase synthesis of thymosin alpha 1, non-peptide impurity and detection method |
| CN103163233B (en) * | 2012-07-12 | 2014-06-25 | 海南合瑞制药股份有限公司 | Deletion peptide and detection method for peptide phase 28 of solid-phase synthesis thymulin alpha1 |
| CN103163235B (en) * | 2012-07-12 | 2014-09-24 | 海南合瑞制药股份有限公司 | Deletion peptide and detection method for peptide phase 16 of solid-phase synthesis thymulin alpha1 |
| CN103163236B (en) * | 2012-07-12 | 2014-09-24 | 海南合瑞制药股份有限公司 | Method for identifying whether purified thymosin alpha 1 contains deletion peptide or not |
| WO2020077927A1 (en) * | 2018-10-14 | 2020-04-23 | 深圳市健元医药科技有限公司 | Post-processing method for gonadotropin-releasing hormone antagonist |
| CN109705206A (en) * | 2019-03-12 | 2019-05-03 | 江苏诺泰澳赛诺生物制药股份有限公司 | A kind of purification process and purposes of thymalfasin |
| CN109705206B (en) * | 2019-03-12 | 2021-12-24 | 江苏诺泰澳赛诺生物制药股份有限公司 | Purification method and application of thymalfasin |
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Application publication date: 20120530 |