CN101597319A - A kind of purification of Recombinant Filgrastim's method - Google Patents
A kind of purification of Recombinant Filgrastim's method Download PDFInfo
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- CN101597319A CN101597319A CNA2008100646530A CN200810064653A CN101597319A CN 101597319 A CN101597319 A CN 101597319A CN A2008100646530 A CNA2008100646530 A CN A2008100646530A CN 200810064653 A CN200810064653 A CN 200810064653A CN 101597319 A CN101597319 A CN 101597319A
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Abstract
The present invention relates to a kind of purification of Recombinant Filgrastim's method.The present invention first with the fine separation step application of reverse phase filler in the purifying process of recombinant methionyl human G-CSF.Compare with prior art, the present invention adopts reverse phase filler fine separation recombinant methionyl human G-CSF first, and electrophoresis purity brings up to 99% by 90%, and HPLC purity brings up to 99% by 90%, and it is than living by 6.1 * 10
7U/mg brings up to 2.3 * 10
8U/mg.Adopt the purifying process of recombinant methionyl human G-CSF of the present invention, purity, the ratio that can significantly improve recombinant methionyl human G-CSF are alive, stable, will effectively reduce its side effect in clinical.
Description
Technical field
The present invention relates to field of biological pharmacy, relate in particular to the proteic purifying of medicinal recombinant human granulocyte colony-stimulating factor, particularly the reverse phase filler chromatography method is at the purification process of recombinant methionyl human G-CSF.
Background technology
Granulocyte colony-stimulating factor (granulocyte colony-stimulating factor, the abbreviation recombinant methionyl human G-CSF) has the effect that promotes the differentiation of granulocyte hemopoietic stem cell proliferation and strengthen ripe cell function, promote during for bone marrow transplantation that neutrophilic leukocyte increases, the serious neutrophil leucocyte deficiency disease that causes during cancer chemotherapy, and the neutrocyte deficiency disease that aplastic anemia is followed all has tangible curative effect, but its natural product source is very limited, and the biologic activity of engineered reorganization recombinant methionyl human G-CSF is to natural similar, and can be mass-produced.
Recombinant methionyl human G-CSF is mainly derived from scavenger cell, endotheliocyte and fibroblast.Precursor cell survival, propagation and the differentiation of the stimulation neutrophilic leukocyte system of energy high degree of specificity, the while also is the functional activation factor of sophisticated neutrophilic leukocyte.Recombinant methionyl human G-CSF is a glycoprotein, and its molecular weight is 19.6KD, and iso-electric point contains 5 cysteine residues for being about 6.0 in the molecule, can form two disulfide linkage.The assignment of genes gene mapping is at karyomit(e) 17, and its gene contains 5 exons and 4 introns, now successfully isolates two cDNA, encode respectively 177 and 174 amino acid whose protein.The recombinant methionyl human G-CSF of two kinds of forms of analysis revealed all has biological activity, has institute's difference but use.
1993, people such as Qu Chengkui at first cloned people's recombinant methionyl human G-CSF cDNA at home, and obtained to express in intestinal bacteria.Because the working ability after the translations such as procaryotic cell expression system shortage glycosylation, the recombinant methionyl human G-CSF of producing can partly be degraded in vivo and be reduced drug effect, so the researchist has carried out the structure of recombinant methionyl human G-CSF gene in the eukaryotic cell expression system, all successfully realized the expression of recombinant methionyl human G-CSF.For the output that makes recombinant methionyl human G-CSF has further raising, the researchist has also carried out the research with recombinant methionyl human G-CSF and other cytokine amalgamation and expression, the acquisition of people's successes such as Cui Libin the solubility expression of recombinant methionyl human G-CSF-sulphur hydrogen reduction fusion protein, expression level is 41% of total cell soluble protein.
At present, the method for relevant recombinant methionyl human G-CSF separation and purification oneself many relevant reports are arranged.At present, adopt " ion-exchange chromatography one exclusion chromatography " method of purification to come the purification of Recombinant Filgrastim mostly.The purification of Recombinant Filgrastim's of elder generation inclusion body can obtain the inclusion body that content is 90% recombinant methionyl human G-CSF, carries out a renaturation and a step ion exchange chromatography then, can make final purity of protein reach 95%, and the rate of recovery is 35%.By " ion-exchange chromatography one hydrophobic chromatography " purification of Recombinant Filgrastim, can reach 6.1 * 10 than work
7U/mg, purity of protein are 98%, and the rate of recovery is 13.2%.Adopt the dilution method recombinant protein, be purified to purity 95% through SP-seharose FF positively charged ion chromatography afterwards, the ratio work of recombinant methionyl human G-CSF can reach 2.3 * 10
8U/mg.
(reverse-phase liquid chromato2graphy is the most widely used a kind of pattern during present liquid chromatography is separated RPLC) to reversed-phase liquid chromatography, is characterized in that the polarity of stationary phase compares weak mobile phase.RPLC compare with other chromatographic processes have resolving power and rate of recovery height, good reproducibility, advantage such as easy and simple to handle.Because RPLC can use volatility system such as water-soluble trifluoroacetic acid (TFA), acetonitrile (ACN) etc., purified product needn't carry out wash-out, has therefore simplified operation steps greatly.In addition, in RPLC, unfolding more or less can take place during by chromatographic column in protein molecule, inner some hydrophobic residue exposes and interacts with stationary phase, thereby show the selectivity different with other chromatograms and electrophoresis method, the information that provides additive method not provide, this becomes the another favorable factor of RPLC in protein separation is analyzed.Therefore, RPLC has become widely used a kind of clastotype at present, generally is used for proteinic compartment analysis.Chlenov etc. have carried out purifying with RPLC to thyrotropin, and Chertov etc. have carried out the RPLC purity analysis to the T-lymphocyte chemotactic factor (LCF).
The problem that traditional method renaturation and purification of Recombinant Filgrastim mainly run into is: the hydrophobicity of recombinant methionyl human G-CSF is very strong, when dilution refolding, a large amount of protein aggregation precipitations, a spot of albumen renaturation in solution is only arranged, and then could continue separated purifying.These characteristics at recombinant methionyl human G-CSF, the present invention adopts the reverse-phase chromatography filler that recombinant methionyl human G-CSF liquid has been carried out purifying, not only make the purity of recombinant methionyl human G-CSF and reach satisfied result, and simplified operation steps greatly, shortened the production cycle than living.
Summary of the invention
The present invention can solve the deficiency of existing recombinant methionyl human G-CSF purification technique, be innovation, increase substantially purity of protein after the recombinant methionyl human G-CSF separation and purification, than living and stable to engineered protein separation and purification means.
The technical scheme that technical problem adopted that the present invention solves is to have adopted the fine separation purification step of reverse phase filler in purifying process.Comprise step:
A, engineering bacteria is carried out fermentation culture;
B, fermentation culture gained thalline is carried out the separation and the washing of inclusion body, obtain the crude product inclusion body;
C, the crude product inclusion body is carried out renaturation, obtain renaturation product;
D, renaturation product is carried out the ion column chromatography handle, obtain ion column chromatography product;
E, ion column chromatography product carry out fine separation, adopt the reverse phase filler column chromatography to handle, and obtain albumen.
Compare with prior art, adopt the reverse phase filler fine separation of this patent, the recombinant methionyl human G-CSF electrophoresis purity brings up to 99% by 90%, and HPLC purity brings up to 99% by 90%, and it is than living by 6.1 * 10
7U/mg brings up to 2.3 * 10
8U/mg.As seen, adopt the purifying process of recombinant methionyl human G-CSF of the present invention, purity, the ratio that can significantly improve recombinant methionyl human G-CSF are alive, stable.Be reduced in the side effect in clinical.
Embodiment
By the following examples, experimental example describes content of the present invention in detail, but these embodiment are not construed as limiting the invention.
Embodiment 1
Use half preparation type MKF-RP post.
Sample: cationic layer division thing 200ml, protein concentration 2mg/ml.
Preparation elutriant A is for containing 50mmolNaCl and 20mmol Tris-HCl damping fluid (pH 8), and elutriant B is for containing 50mmolNaCl solution 20mmol Tris-HCl 60% acetonitrile (pH 8).
With elutriant A wash-out, use gradient elution 15%-90% elutriant B wash-out more earlier, 10-55 minute collection sample peak.
The sample that is collected is removed organic substance through low-temperature evaporation, and dialysis is kept under 4 ℃ of conditions after concentrating then.
Embodiment 2
Use XK 50/30 post, with the anti-phase preparative liquid chromatography filler dress of SBC MCL GEL type post.
Sample: anion chromatography product 500ml, protein concentration 2.2mg/ml.
Elutriant A is 20% phosphate buffered saline buffer, and elutriant B is 30% methanol solution that contains 0.02%tween80, and flow velocity is 15ml/min.
Gradient elution at 10-80 minute 20%-75% elutriant B wash-out, is collected the sample peak.When collecting sample, collect middle portion.The sample that is collected is removed organic substance through low-temperature evaporation, and dialysis is kept under 4 ℃ of conditions after concentrating then.
Embodiment 3
Use Butyl-Sepharose 4Fast Flow, the XK50H16 chromatography column.
Sample: anion chromatography product 500ml, protein concentration 1.5mg/ml.Add ammonium sulfate to 0.8M.
Balance liquid: 20mmol/ml Tris-HCl, PH8.0,0.8M ammonium sulfate
Pre-elutriant: 20mmol/ml Tris-HCl, PH8.0,0.5M ammonium sulfate
Elutriant: 20mmol/ml Tris-HCl, PH8.0,0.2M ammonium sulfate
Collect elutriant wash-out part, the dialysis desalination promptly.
Under 4 ℃ of conditions the sample of embodiment 1,2,3 is stablized investigation, it is as follows to investigate the result:
0 month
March
June
December
Above data declaration, embodiment 1,2 samples that obtain by reverse stuffing means are than embodiment 3, and electrophoresis purity and HPLC purity all greater than 97%, reached 2.0 * 10 than work in 12 months
8More than the U/mg, other every indexs also meet the pharmacopeia requirement, and embodiment 1,2 samples can be placed 12 months under 4 ℃ of conditions, and embodiment 3 samples occur reducing the phenomenon that purity of protein descends than living when investigating 6 months.Illustrate by reverse stuffing means acquisition recombinant methionyl human G-CSF albumen more stable.
Claims (5)
1, a kind of purification of Recombinant Filgrastim's processing method.It is characterized in that having adopted the reverse phase filler chromatographic separation.
2, the sample requirement before the reverse phase filler chromatographic separation according to claim 1 is characterized in that through preliminary separation and purification.
3, initial gross separation purifying according to claim 2 is characterized in that handling through negatively charged ion or cation seperation column chromatography.
4, reverse phase filler post according to claim 1, its feature is adopting prepackage type post and preparation type post.
5, reverse phase filler chromatography according to claim 1, its feature is adopting reverse-phase chromatography prepacked column or reverse phase filler prepacked column.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103014100A (en) * | 2012-12-25 | 2013-04-03 | 哈药集团生物工程有限公司 | Purifying method for recombinant human granulocyte stimulating factor |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103014100A (en) * | 2012-12-25 | 2013-04-03 | 哈药集团生物工程有限公司 | Purifying method for recombinant human granulocyte stimulating factor |
| CN103014100B (en) * | 2012-12-25 | 2014-10-01 | 哈药集团生物工程有限公司 | Purifying method for recombinant human granulocyte stimulating factor |
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Application publication date: 20091209 |