CN100354301C - Novel large-scale separation and preparation technology of chemically synthesized polypeptide - Google Patents
Novel large-scale separation and preparation technology of chemically synthesized polypeptide Download PDFInfo
- Publication number
- CN100354301C CN100354301C CNB2005100947708A CN200510094770A CN100354301C CN 100354301 C CN100354301 C CN 100354301C CN B2005100947708 A CNB2005100947708 A CN B2005100947708A CN 200510094770 A CN200510094770 A CN 200510094770A CN 100354301 C CN100354301 C CN 100354301C
- Authority
- CN
- China
- Prior art keywords
- polypeptide
- chemically synthesized
- synthesized polypeptide
- solution
- isolating method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 113
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 83
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 81
- 238000000926 separation method Methods 0.000 title claims abstract description 33
- 238000005516 engineering process Methods 0.000 title abstract description 23
- 238000002360 preparation method Methods 0.000 title abstract description 6
- 238000001728 nano-filtration Methods 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 17
- 238000005342 ion exchange Methods 0.000 claims abstract description 14
- 239000012528 membrane Substances 0.000 claims abstract description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims abstract description 6
- 238000004255 ion exchange chromatography Methods 0.000 claims abstract description 6
- 239000003456 ion exchange resin Substances 0.000 claims abstract description 6
- 229920003303 ion-exchange polymer Polymers 0.000 claims abstract description 6
- 230000010355 oscillation Effects 0.000 claims abstract description 6
- 230000002209 hydrophobic effect Effects 0.000 claims abstract description 4
- 239000007787 solid Substances 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 21
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 8
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
- 239000001166 ammonium sulphate Substances 0.000 claims description 5
- 239000003480 eluent Substances 0.000 claims description 5
- 238000001914 filtration Methods 0.000 claims description 5
- MAZPIOPHRNWBNG-UHFFFAOYSA-N [Cl-].Cl[NH3+].[Na] Chemical compound [Cl-].Cl[NH3+].[Na] MAZPIOPHRNWBNG-UHFFFAOYSA-N 0.000 claims description 4
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 claims description 4
- 239000012266 salt solution Substances 0.000 claims description 4
- -1 sulfopropyl Chemical group 0.000 claims description 4
- ZMANZCXQSJIPKH-UHFFFAOYSA-N triethylamine Natural products CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 4
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- 239000004793 Polystyrene Substances 0.000 claims description 3
- 150000002148 esters Chemical class 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- 229920002401 polyacrylamide Polymers 0.000 claims description 3
- 229920002223 polystyrene Polymers 0.000 claims description 3
- WIHIUTUAHOZVLE-UHFFFAOYSA-N 1,3-diethoxypropan-2-ol Chemical compound CCOCC(O)COCC WIHIUTUAHOZVLE-UHFFFAOYSA-N 0.000 claims description 2
- 238000010521 absorption reaction Methods 0.000 claims description 2
- 230000003993 interaction Effects 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 230000008901 benefit Effects 0.000 abstract description 4
- 239000012535 impurity Substances 0.000 abstract description 4
- 238000005406 washing Methods 0.000 abstract description 4
- 238000011033 desalting Methods 0.000 abstract 1
- 238000004108 freeze drying Methods 0.000 abstract 1
- 231100000614 poison Toxicity 0.000 abstract 1
- 230000007096 poisonous effect Effects 0.000 abstract 1
- 238000001179 sorption measurement Methods 0.000 abstract 1
- 239000000047 product Substances 0.000 description 12
- 238000000746 purification Methods 0.000 description 10
- 239000003814 drug Substances 0.000 description 7
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 5
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 4
- 108010000817 Leuprolide Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 238000001641 gel filtration chromatography Methods 0.000 description 4
- GFIJNRVAKGFPGQ-LIJARHBVSA-N leuprolide Chemical compound CCNC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)CC1=CC=C(O)C=C1 GFIJNRVAKGFPGQ-LIJARHBVSA-N 0.000 description 4
- 229960004338 leuprorelin Drugs 0.000 description 4
- 239000007790 solid phase Substances 0.000 description 4
- 238000010532 solid phase synthesis reaction Methods 0.000 description 4
- 102000007501 Thymosin Human genes 0.000 description 3
- 108010046075 Thymosin Proteins 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000006227 byproduct Substances 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 210000003746 feather Anatomy 0.000 description 3
- 239000012982 microporous membrane Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 238000003786 synthesis reaction Methods 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
- 229920002307 Dextran Polymers 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 235000019270 ammonium chloride Nutrition 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 239000002547 new drug Substances 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000004304 visual acuity Effects 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102100025064 Cellular tumor antigen p53 Human genes 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 101000721661 Homo sapiens Cellular tumor antigen p53 Proteins 0.000 description 1
- 229920002684 Sepharose Chemical group 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 229940088623 biologically active substance Drugs 0.000 description 1
- 238000005341 cation exchange Methods 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 125000002228 disulfide group Chemical group 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000006340 racemization Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 238000002444 silanisation Methods 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The present invention discloses a novel large-scale separation and preparation technology for chemically synthesized polypeptide. In the technology, chemically synthesized polypeptide is separated by adopting a separation method combining normal-pressure or low-voltage ion exchange chromatography and nanofilteration. In a specific method, when coarse peptide dissolves in water, ultrasonic oscillation is adopted to assist the coarse peptide to dissolve in water, and solid insoluble matter is removed by using a microporous filter membrane of 0.22 mu m to obtain clear coarse peptide solution; hydrophobic ionexchange resin is used as a separating medium, and a ratio of height to diameter is 5:1 to 20:1; the coarse peptide solution is processed through column attachment to be processed through ion-exchange adsorption, impurity washing, and elutriation; a nanofiltration membrane with different catch molecular weight is selected according to the molecular weight of separated polypeptide to carry out nanofiltration, concentration and desalting to ion exchange elutriant; finally, the concentrated polypeptide solution is processed through freeze drying. The technology of the present invention avoids using expensive equipment and elutriating by using poisonous organic solution. The present invention has the advantages of large carrying capacity of the separating medium, little equipment investment, easy scale production and low production cost.
Description
Technical field
The invention belongs to the biological products manufacture field, relate to a kind of novel large-scale separation and preparation technology chemically synthesized polypeptide.
Technical background
Polypeptide relates to the biologically active substance of various cell functions in the organism.The synthetic polypeptide of manual method is T﹠B, is accompanied by the develop rapidly of molecular biology, Measurement for Biochemistry, and the research of polypeptide has obtained surprising epoch-making progress.It is found that the polypeptide that is present in organism has tens thousand of kinds, and find that all cells can both synthesize polypeptide.Simultaneously, nearly all cell also all is subjected to polypeptides for modulating, and it relates to every field such as hormone, nerve, cell growth and reproduction.At present, the application of polypeptide in medicine mainly concentrates on polypeptide vaccine, tumor protein p53, antiviral polypeptide, polypeptide targeted drug, cytokine simulating peptide, antibacterial active peptide, is used for polypeptide and diagnosis polypeptide or the like of cardiovascular disorder, and the application of peptide medicament has related to 24 class clinical indications.Along with modern technologies are maked rapid progress, polypeptide is not only as the new drug development target, simultaneously also as the screening target of developing other medicine.According to incompletely statistics, the annual growth of polypeptide drug reaches 19%.Polypeptide drugs are compared with small-molecule drug, and it has advantages such as mechanism is clear and definite, side effect is little.A large amount of discovers, nearly all virus disease and many difficult diseases can prevent and treat with polypeptide, and therefore, the polypeptide technology will be accomplished something at aspects such as prevention and treatment epidemic infectious diseases and major diseases.
From the seventies in 20th century, along with the development of the solid phase synthesis of polypeptide and high performance liquid phase purifying, analytical technology, polypeptide more and more causes people's interest as a potential new drug.At present, artificial synthetic polypeptide is mainly based on solid phase synthesis.But because in the solid phase synthesis process, problems such as the various side reactions of appearance, racemization make that synthetic this polypeptide is a kind of thick product of low-purity, these impurity comprise: 1. diastereoisomeric (racemize) polypeptide; 2. lack (not exclusively) peptide; 3. peptide ruptures; 4. byproduct of reaction; 5. remove the acid amides polypeptide; 6. the formed by product of incomplete deprotection of amino acid side chain; 7. oxidation peptide; 8. the product of disulfide exchange; 9. oligopolymer and/or polymkeric substance; 10. used toxic agent and solvent in synthetic.Must just can obtain high purity product through purifying, separation and purification problem thus is the 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 separation means commonly used has three kinds: gel filtration chromatography, ion-exchange chromatography, RPLC.Gel filtration chromatography is many to be cross-linked into reticulated structure as medium with linking agent with dextran and agarose, size differences, shape difference according to separated object separate, be mainly used in the desalination of polypeptide, gel filtration chromatography has the medium neutral, the separation condition gentleness, rate of recovery height, characteristics such as biological activity is good; The separating medium of ion-exchange chromatography is to be bonded to dextran or sepharose carrier by diethylamine ethyl (DEAE), QAE (QAE), carboxymethyl (CM), phosphate (P), sulfopropyl (SP) plasma cation exchange groups and to constitute, difference according to electrostatic force between charged separated object and the ion-exchanger is carried out lock out operation, ion-exchange chromatography has higher separation capacity, the resolving power height, be easy to characteristics such as amplification, be widely used in the separation and purification of biomacromolecule.The medium of RPLC be with silica gel as carrier, by Silanization reaction at silica gel surface bond C
4, C
8, C
18Non-polar molecule layers such as alkyl or phenyl carry out separation and purification according to the hydrophobic difference of separated object, and RPLC has good separating effect, characteristics such as resolving power height, rate of recovery height.At present, the main preparation method of the separation of chemical synthesising peptide is a RPLC, and it is used for amplifying the synthetic polypeptide of separation, be difficult for mass-producing, and separation costs is too high.Major cause, on the one hand, because anti-phase supported carrier amount is low, cost height own, scale zoom facility precision prescribed height, investment are big, and become one of difficult point that the solid-phase polypeptide separation and purification amplifies; On the other hand, solid-phase polypeptide separation and purification means are single at present, and consumption of organic solvent is big, the energy consumption height.The present domestic extensive isolating industrialization example of chemically synthesized polypeptide that still do not have.
It is flourish that external polypeptide is made the field, formed certain industrialized scale.There are Bachem company, Novabiochem company, Anaspec﹠amp in famous enterprise; ACT company, Anaspec company and American Peptide company etc., wherein with Bachem company size maximum, adopt the synthetic polypeptide of solid phase synthesis technique and liquid phase synthetic technology, can produce from milligram level polypeptide to hundreds of feather weight polypeptide and the relevant product of all kinds of and polypeptide of tonne intermediate, maximum single batch of production separation and purification ability reaches kilogram more than the rank, and its separation and purification means mainly are based on RPLC.Though the synthetic enterprise of domestic polypeptide has much, polypeptide synthesis capability maximum only reaches feather weight, and the separation scale that matches with it also rests on gram level level.From in general, domestic no matter aspect synthesis capability, or separating power aspect and external gap are quite huge.With regard to local, domestic existing separation of level also can't adapt with synthesis capability, has become one of bottleneck of the synthetic development of restriction solid-phase polypeptide.
Along with the develop rapidly of membrane technique, adopting membrane technique to substitute gel filtration chromatography has become possibility.If adopt small molecules separation and purification means, effectively separate target polypeptides and impurity in the solid-phase synthetic peptide, realize the production domesticization of carrier of separating, reduce process loss, reduce the polypeptide cost significantly, promote the development of China's polypeptide drugs, realize that the high efficiency, low cost polypeptide is synthetic, this will bring remarkable economic efficiency and social value.
Summary of the invention
The objective of the invention is to overcome separating and purifying technology in the present chemically synthesized polypeptide the cost height, be difficult for the defective of mass-producing, provide a kind of ion-exchange separated the chemically synthesized polypeptide novel separation and purification process that combines with nanofiltration membrane (Nanofiltration Membrane).
The present invention is based upon on the small molecular orientation separating thought, its characteristics are to utilize in the solution between the target product and coexistent impurity the difference in physics, chemistry and biological property, make its in lock out operation, have different rate of mass transfer and (or) equilibrium state, thereby realize the isolating purpose of target compound.
According to technical scheme of the present invention, the one of ordinary skilled in the art need not creative work just can implement the present invention.Especially those skilled in the art understand each process that should carry out, can obtain target polypeptides.
Purpose of the present invention can realize by following measure:
A kind of to the extensive isolating technology of chemically synthesized polypeptide, the synthetic polypeptide of the separation method separation chemistry that this technology adopts normal pressure or low-voltage ion exchange chromatography to combine with nanofiltration, concrete grammar is: when thick peptide is water-soluble, adopt the ultra-sonic oscillation assist in dissolving, remove solid shape insolubles with 0.22 μ m millipore filtration again, obtain clarifying thick peptide solution; Utilize hydrophobicity ion exchange resin as separating medium, aspect ratio is 5: 1～20: 1; Thick peptide solution upper prop is carried out ion-exchange absorption, adopt suitable assorted and the wash-out washed again; According to the molecular weight size of separated polypeptide, select the nanofiltration membrane of PSPP, ion-exchanging eluent is carried out the nanofiltration concentrating and desalinating; At last, spissated polypeptide solution is obtained the polypeptides freeze-dry powder of purity more than 95% through lyophilize.
Described to the extensive isolating technology of chemically synthesized polypeptide, when wherein thick peptide was water-soluble, the mass volume ratio of thick peptide and water was 1: 5～1: 15.
Described to the extensive isolating technology of chemically synthesized polypeptide, wherein separating medium with polyacrylic ester, polyacrylamide or polystyrene as the hydrophobic interaction skeleton.
Described to the extensive isolating technology of chemically synthesized polypeptide, wherein the active group of separating medium is diethylamine ethyl, diethylin, triethylamine ethyl, QAE, sulfonic group, sulfopropyl, phosphate or carboxymethyl.
Described to the extensive isolating technology of chemically synthesized polypeptide, wherein adopt the inorganic salt solution of 0.001M～0.2M to wash assorted.
Described to the extensive isolating technology of chemically synthesized polypeptide, wherein inorganic salt are sodium-chlor, ammonium chloride or the ammoniumsulphate soln of pH2～14.
Described to the extensive isolating technology of chemically synthesized polypeptide, wherein adopt the inorganic salt solution of 0.5M～2M to carry out wash-out.
Described to the extensive isolating technology of chemically synthesized polypeptide, wherein inorganic salt are sodium-chlor, ammonium chloride or the ammoniumsulphate soln of pH2～14.
Described to the extensive isolating technology of chemically synthesized polypeptide, wherein the nanofiltration membrane molecular weight cut-off of Cai Yonging is 150～1000 dalton, carries out concentrating and desalinating.
The present invention has following advantage compared to existing technology:
The present invention breaks through the inflexible and medelling of domestic and international colleague to chemically synthesized polypeptide separation and purification mode, ion-exchange is combined with nanofiltration membrane separation, make that separating and purifying technology of the present invention is simple and easy to do, effect is good, not only its facility investment, running cost are cheap, can realize the separation of hectogram level, feather weight chemically synthesized polypeptide, prior art and the present invention's contrast, as shown in table 1.All having obtained fabulous result aspect product yield and the quality product, guaranteed the high-quality of product by products obtained therefrom of the present invention, separation costs of the present invention has only about 7% of prior art, has reduced the separation costs of chemically synthesized polypeptide significantly.
Table 1 prior art and contrast of the present invention
| Project | Prior art | The present invention |
| Single cover preparation equipment cost | 160,000～600,000 yuan | 20,000 yuan |
| 10 gram level chromatographic column costs | 200,000～400,000 yuan | 5000 yuan |
| Column pressure | Middle and high pressure | Normal pressure, low pressure |
| The separating medium median size | 5μm | 0.7mm |
| Single batch of running cost | 2000～4000 yuan | About 150 yuan |
| Scale is amplified | Be difficult for amplifying, and the cost height | Be easy to amplify, and cost is low |
Other advantages of the present invention will further be set forth in the illustrated example below.
Embodiment
The invention will be further elaborated below in conjunction with embodiment.Embodiment is to be the unrestricted the present invention of explanation.Any those of ordinary skill can be understood these embodiments and not limit the present invention in any way in this area, can make suitable modification and without prejudice to essence of the present invention with depart from scope of the present invention.
Embodiment 1
1.2 kilogram thick peptide of thymopeptide-5 and pure water (mass volume ratio is 1: 5) are mixed, ultra-sonic oscillation under the room temperature condition, and, obtain clarifying thick peptide solution with 0.22 μ m filtering with microporous membrane.6 kilograms of sulfopropyl polystyrene ion-exchange resin dress posts (aspect ratio is 7: 1) are standby, then thick peptide settled solution is adsorbed with 10ml/min flow velocity upper prop.With pH11,0.1M ammonium sulfate solution, 20ml/min flow velocity drip washing resin does not occur to there being assorted peak; Then with 1.0M sodium chloride solution (pH=10～12), 10ml/min carries out wash-out, and ion exchange process adopts the 275nm wavelength to detect, and working pressure maintains below the 0.2MPa, collects the thymopeptide-5 ion-exchanging eluent; With molecular weight cut-off is 150 daltonian nanofiltration membrane, under normal temperature condition, elutriant is carried out the nanofiltration concentrating and desalinating, the nanofiltration operational condition: pressure is 2.0MPa, and flow is 3000ml/min; With the concentrated solution lyophilize, obtaining purity is the pure product of 99.1% thymopeptide-5 at last.
Embodiment 2
150 gram thick peptides of thymosin and pure water (mass volume ratio is 1: 15) are mixed, ultra-sonic oscillation under the room temperature condition, and, obtain clarifying thick peptide solution with 0.22 μ m filtering with microporous membrane.700 gram sulfonic group polyacrylamide ion exchange resin dress posts (aspect ratio is 12: 1) are standby, then thick peptide settled solution is adsorbed with 1.5ml/min flow velocity upper prop.With pH5,0.15M sodium chloride solution, 2ml/min flow velocity drip washing resin does not occur to there being assorted peak; Then with 1.4M ammonium chloride solution (pH=7～10), 1.5ml/min carries out wash-out, and ion exchange process adopts under condition of normal pressure, and the 214nm wavelength detects; Collect the thymosin ion-exchanging eluent; With molecular weight cut-off is 1000 daltonian nanofiltration membrane, under normal temperature condition, elutriant is carried out the nanofiltration concentrating and desalinating, the nanofiltration operational condition: pressure is 1.5MPa, and flow is 1000ml/min; With the concentrated solution lyophilize, obtaining purity is the pure product of 97.8% thymosin at last.
Embodiment 3
500 gram thick peptides of Leuprolide and pure water (mass volume ratio is 1: 10) are mixed, ultra-sonic oscillation under the room temperature condition, and, obtain clarifying thick peptide solution with 0.22 μ m filtering with microporous membrane.2500 gram diethylamine ethyl polyacrylic ester ion exchange resin dress posts (aspect ratio is 15: 1) are standby, then the thick peptide settled solution of Leuprolide is adsorbed with 2ml/min flow velocity upper prop.With pH9 0.2M aqueous ammonium chloride solution, 4ml/min flow velocity drip washing resin does not occur to there being assorted peak.Then with 2.0M ammoniumsulphate soln (pH=9～13), the 1ml/min flow velocity carries out wash-out, and ion exchange process adopts the 220nm wavelength to detect, and working pressure maintains below the 0.1MPa; Collecting the Leuprolide ion-exchanging eluent, is 300 daltonian nanofiltration membrane with molecular weight cut-off, and under normal temperature condition, elutriant is carried out the nanofiltration concentrating and desalinating, the nanofiltration operational condition: pressure is 0.6MPa, and flow is 800ml/min; With the concentrated solution lyophilize, obtaining purity is the pure product of 98.3% Leuprolide at last.
Claims (9)
1, a kind of to the extensive isolating method of chemically synthesized polypeptide, it is characterized in that the synthetic polypeptide of the separation method separation chemistry that adopts normal pressure or low-voltage ion exchange chromatography to combine with nanofiltration, concrete grammar is: when thick peptide is water-soluble, adopt the ultra-sonic oscillation assist in dissolving, remove solid shape insolubles with 0.22 μ m millipore filtration again, obtain clarifying thick peptide solution; Utilize hydrophobicity ion exchange resin as separating medium, aspect ratio is 5: 1～20: 1; Thick peptide solution upper prop is carried out ion-exchange absorption, adopt suitable assorted and the wash-out washed again; According to the molecular weight size of separated polypeptide, select the nanofiltration membrane of PSPP, ion-exchanging eluent is carried out the nanofiltration concentrating and desalinating; At last, spissated polypeptide solution is obtained the polypeptides freeze-dry powder of purity more than 95% through lyophilize.
2, according to claim 1 to the extensive isolating method of chemically synthesized polypeptide, when it is characterized in that thick peptide is water-soluble, the mass volume ratio of thick peptide and water is 1: 5～1: 15.
3, according to claim 1 to the extensive isolating method of chemically synthesized polypeptide, it is characterized in that separating medium with polyacrylic ester, polyacrylamide or polystyrene as the hydrophobic interaction skeleton.
4, according to claim 1 to the extensive isolating method of chemically synthesized polypeptide, the active group that it is characterized in that separating medium is diethylamine ethyl, diethylin, triethylamine ethyl, QAE, sulfonic group, sulfopropyl, phosphate or carboxymethyl.
5, according to claim 1 to the extensive isolating method of chemically synthesized polypeptide, it is characterized in that adopting the inorganic salt solution of 0.001M～0.2M to wash assorted.
6, according to claim 5 to the extensive isolating method of chemically synthesized polypeptide, it is characterized in that inorganic salt are sodium-chlor, ammonium chloride or the ammoniumsulphate soln of pH2～14.
7, according to claim 1 to the extensive isolating method of chemically synthesized polypeptide, it is characterized in that adopting the inorganic salt solution of 0.5M～2M to carry out wash-out.
8, according to claim 7 to the extensive isolating method of chemically synthesized polypeptide, it is characterized in that inorganic salt are sodium-chlor, ammonium chloride or the ammoniumsulphate soln of pH2～14.
9, according to claim 1 to the extensive isolating method of chemically synthesized polypeptide, it is characterized in that the nanofiltration membrane molecular weight cut-off that adopts is 150～1000 dalton, carries out concentrating and desalinating.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100947708A CN100354301C (en) | 2005-10-13 | 2005-10-13 | Novel large-scale separation and preparation technology of chemically synthesized polypeptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100947708A CN100354301C (en) | 2005-10-13 | 2005-10-13 | Novel large-scale separation and preparation technology of chemically synthesized polypeptide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1800203A CN1800203A (en) | 2006-07-12 |
| CN100354301C true CN100354301C (en) | 2007-12-12 |
Family
ID=36810447
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100947708A Expired - Fee Related CN100354301C (en) | 2005-10-13 | 2005-10-13 | Novel large-scale separation and preparation technology of chemically synthesized polypeptide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100354301C (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003016348A2 (en) * | 2001-08-14 | 2003-02-27 | Statens Serum Institut | A purification process for large scale production of gc-globulin, product obtained thereby and their use in medicine |
| FR2857978A1 (en) * | 2003-07-25 | 2005-01-28 | Simer Laboratoires Science Et | Preparing a peptide extract of Spirulina, useful in nutraceutical and cosmetic compositions for e.g. controlling aging of the skin, comprises extraction of lipids then enzymatic hydrolysis |
| WO2005014648A1 (en) * | 2003-08-12 | 2005-02-17 | Octapharma Ag | Process for preparing an alpha-1-antitrypsin solution |
-
2005
- 2005-10-13 CN CNB2005100947708A patent/CN100354301C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003016348A2 (en) * | 2001-08-14 | 2003-02-27 | Statens Serum Institut | A purification process for large scale production of gc-globulin, product obtained thereby and their use in medicine |
| FR2857978A1 (en) * | 2003-07-25 | 2005-01-28 | Simer Laboratoires Science Et | Preparing a peptide extract of Spirulina, useful in nutraceutical and cosmetic compositions for e.g. controlling aging of the skin, comprises extraction of lipids then enzymatic hydrolysis |
| WO2005014648A1 (en) * | 2003-08-12 | 2005-02-17 | Octapharma Ag | Process for preparing an alpha-1-antitrypsin solution |
Non-Patent Citations (2)
| Title |
|---|
| 离子交换法分离固相合成胸腺五肽 陈永森等.离子交换与吸附，第21卷第3期 2005 * |
| 纳滤膜在工业上的应用 宋玉军等.化工新型材料，第27卷第5期 1999 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1800203A (en) | 2006-07-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5948343B2 (en) | Method for purifying human serum albumin from seeds of transgenic rice | |
| PT1571208E (en) | Novel factor ix purification methods | |
| CN106146652A (en) | A kind of method for extraction and purification of middle phycocyanin of delivering vegetables | |
| CN103421079A (en) | Modification method of polyethyleneglycol of protein | |
| CN108358989A (en) | A method of isolating and purifying cytidine from microbial fermentation solution | |
| CN104927044A (en) | Preparation method of high-purity polyethylene glycol aldehyde derivative | |
| CN103880945A (en) | Method for preparing high-purity thymalfasin | |
| JPS6356501A (en) | Cellulose gel having biochemical affinity and production thereof | |
| CN102408468B (en) | Argatroban compound and preparation method thereof | |
| CN100354301C (en) | Novel large-scale separation and preparation technology of chemically synthesized polypeptide | |
| JPS59231097A (en) | Manufacture of homogeneous human immunological interferon subtype 26k and 21k | |
| CN107629115A (en) | A kind of purification process of Te Lawan stars | |
| CN107827977A (en) | A kind of method based on ion-exchange resin purification UTI | |
| CN112279895B (en) | Preparation method of chemically synthesized acidic polypeptide | |
| CN107629116A (en) | A kind of purification process of Te Lawan stars | |
| CN104311569B (en) | A kind of method extracted with preliminary purification Fugu ocellatus toxin | |
| CN107082796A (en) | A kind of method of purifying protein zymolyte small molecular polypeptide | |
| CN105837685A (en) | Method for purifying ulinastatin based on anion exchange resin | |
| CN101362792A (en) | Affinity separation polymer of lactoferrin and affinity purification method of lactoferrin | |
| CN115806591B (en) | Purification method of veracat peptide | |
| CN101434640A (en) | Method for modifying genetic engineering protein by using polyethyleneglycol | |
| CN101407538B (en) | Solid phase synthesis method of ZP120 | |
| CN109748954A (en) | A kind of purification process of Ac-D-2Nal-D-4Cpa-D-3Pal-Ser-4Aph(Hor)-D-4Aph(Cbm)-Leu-Lys(iPr)-Pro-D-Ala-NH2 | |
| CN113797905B (en) | Pyridine dicarboxamide bridged bis-beta-cyclodextrin stationary phase and preparation method and application thereof | |
| Pirotta | Ion exchangers in pharmacy, medicine and biochemistry |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20071212 Termination date: 20151013 |
|
| EXPY | Termination of patent right or utility model |