CN103114115B - Method for preparing recombinant human granulocyte colony stimulating factors screened by microorganisms, and medicament composition and preparation thereof - Google Patents
Method for preparing recombinant human granulocyte colony stimulating factors screened by microorganisms, and medicament composition and preparation thereof Download PDFInfo
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- CN103114115B CN103114115B CN201310027678.4A CN201310027678A CN103114115B CN 103114115 B CN103114115 B CN 103114115B CN 201310027678 A CN201310027678 A CN 201310027678A CN 103114115 B CN103114115 B CN 103114115B
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Abstract
The invention relates to the field of bio-pharmaceuticals, and in particular relates to a method for preparing recombinant human granulocyte colony stimulating factors screened by microorganisms. The preparation method comprises the following steps of: selecting engineering bacteria of the recombinant human granulocyte colony stimulating factors to ferment and culture; ultrasonically pulverizing bacteria bodies obtained by fermentation medium; collecting inclusion bodies; washing the inclusion bodies with inclusion body washing solution and centrifuging at a low temperature; adding inclusion body dissolution liquid into the inclusion bodies, standing for 8-12 hours at 4 DEG C and centrifuging to obtain inclusion body extraction liquid; separating the extraction liquid by using a Sephacry1S-200 gel column; collecting a plurality of recombinant human granulocyte colony stimulating factor fractions; and separating by using a SephadexG25 column to obtain the recombinant human granulocyte colony stimulating factor. The invention also relates to a medicament composition and a preparation of the recombinant human granulocyte colony stimulating factors.
Description
Technical field
The present invention relates to field of biological pharmacy, specifically, relate to a kind of method that recombinant human granulocyte colony stimulating growth factor is prepared with microbe to screen, its pharmaceutical composition and preparation.
Background technology
Granulocyte colony-stimulating factor (G-CSF) is a kind of glycoprotein, contains 174 amino acid, and molecular weight is about 20000.Mainly can activated monocyte by intracellular toxin, TNF-α and IFN-γ and scavenger cell produce.G-CSF full length gene 2.5kb, comprises 5 exons and 4 introns, and G-CSF has 5 halfcystines, Cys36 and Cys42, between Cys74 and Cys64, form two pairs of disulfide linkage, Cys17 is unpaired halfcystine, and disulfide linkage is necessary factor for maintaining G-CSF biological function.People and mouse G-CSF have 73% homology on amino acid levels, and have cross one another biologic activity.G-CSF Main Function is in propagation, differentiation and the activation of neutrophil series (lineage) hematopoietic cell.
Macrophage colony stimulating factor of recombinant human granulocyte (rhGM-CSF) acts on hemopoietic progenitor cell, promote its propagation and differentiation, its vital role is to stimulate grain, mononuclear macrophage maturation, promote mature cell to discharge to peripheral blood, and can promote scavenger cell and bite the several functions of acid cell.Clinical leukopenia, treatment marrow hemopoiesis dysfunction and the myelodysplastic syndrome that is mainly used in causing after prevention and treatment tumor radiotherapy or chemotherapy, infection complication that prevention oligoleukocythemia may be potential and make to infect the recovery that the neutrophil leucocyte that causes reduces and accelerate.
The human granulocyte colony stimulating growth factor is a kind of hemopoieticgrowth factor, it stimulating proliferation, the aspects such as function of differentiation and activated cytopenia have important effect; Its molecule comprises a free halfcystine at 17, and contains two pairs of disulfide linkage, i.e. Cys36-Cys42 and Cys64-Cys74, and these two pairs of disulfide linkage are necessary for its activity; Yet the source of hG-CSF is very limited, therefore, in industry, must produce it by engineered method.Conventionally in E.coli, express restructuring hG-CSF(rhG-CSF), it exists with the form of water-fast inclusion body, therefore must dissolve with the denaturing agent of high density, dissolving like this albumen obtaining exists with unfolding state, must carry out renaturation to it, then adopt multistep chromatographic step to carry out purifying to it, but its mass recovery is less than 20%.
For this reason, the present invention proposes the preparation method of the recombinant human granulocyte colony stimulating growth factor that a kind of rate of recovery is high, biological activity is high.
Summary of the invention
Goal of the invention of the present invention has been to propose a kind of method that recombinant human granulocyte colony stimulating growth factor is prepared with microbe to screen.
In order to realize object of the present invention, the technical scheme of employing is:
A preparation method for recombinant methionyl human G-CSF, is characterized in that, described preparation method's step is:
(1) engineering bacteria of recombinant methionyl human G-CSF is carried out to fermentation culture;
(2) fermention medium gained thalline is carried out to ultrasonication, collect inclusion body, with inclusion body washings, wash rear low-temperature centrifugation;
(3) in inclusion body, add solubilization of inclusion bodies liquid, under 4 ℃ of conditions after 8～12 hours, the centrifugal inclusion body extracting solution that obtains;
(4) under 2～8 ℃ of conditions, adopt SephacrylS-200 gel column to carry out separation to extracting solution, collect the recombinant methionyl human G-CSF cut of renaturation;
(5) adopt again SephadexG25 post to carry out separation, obtain people's recombinant human colony-stimulating factor.
The first optimal technical scheme of the present invention is: in step (2), by TE damping fluid washing 1～3 time for the thalline of collecting, then adopt ultrasonication 0.5～1 hour, low-temperature centrifugation, collects inclusion body; Add again inclusion body washings, under 0～4 ℃ of condition, mix 20～60 minutes low-temperature centrifugation.
The second optimal technical scheme of the present invention is: in step (2), described TE damping fluid is: 20mmol/LTris-HCl+1mmol/L EDTA, pH8.0; Inclusion body washings is: 20mmol/L Tris-HCl+1mmol/L EDTA+2mmol/L urea+0.5%Tritonx-100, pH8.0; The volume ratio of inclusion body weight and inclusion body washings is 1:10～20, preferably 1:10.
The 3rd optimal technical scheme of the present invention is: in step (3), described solubilization of inclusion bodies liquid is: 20mmol/LTris-HCl+1mmol/L EDTA+7mmol/L Guanidinium hydrochloride+20mmol/L DTT, pH8.0; Inclusion body quality with the ratio of the volume of solubilization of inclusion bodies liquid is: 1:5～10, preferably 1:10.
The 4th optimal technical scheme of the present invention is: in step (4), described chromatographic condition is:
Moving phase is: 0.15mol/L NaCl+0.05mol/L Tris-HCl+1mmol/L EDTA+2mmol/L urea+0.8mmol/LGSH+0.2mmol/L GSSG, pH7.8;
Flow velocity is 2mL/min;
Check wavelength is 280nm.
The 5th optimal technical scheme of the present invention is: in step (4), described Superdex75 gel column is first used moving phase balance, and then sample introduction carries out separation to inclusion body extracting solution.
The 6th optimal technical scheme of the present invention is: in step (5), it is 4.0～4.5 that people's rhG-CSF cut of the renaturation of collection is adjusted to pH with 10% Glacial acetic acid, and preferably pH is 4.2; Low-temperature centrifugation obtains supernatant liquor in 20～40 minutes, and SephadexG25 post on supernatant liquor, uses elutriant wash-out, collects main peak and carries out separation, obtains people's recombinant human colony-stimulating factor.
The 7th optimal technical scheme of the present invention is: in step (5), it is the sodium-acetate balance of 4.0～4.5 20mmol/L that described SephadexG25 post first adopts pH, and being preferably pH is 4.2.
The 8th optimal technical scheme of the present invention is: in step (5), described elutriant is: the sodium-acetate of sodium-chlor+15mmol/L of 10mmol/L, pH is 4.4.
The invention still further relates to a kind of pharmaceutical composition of the recombinant methionyl human G-CSF being prepared by preparation method of the present invention, consisting of of described pharmaceutical composition: recombinant methionyl human G-CSF 100 weight parts, N.F,USP MANNITOL 5～100 weight parts.The formulation of described pharmaceutical composition is freeze-dried powder or liquid drugs injection; Wherein, when described formulation is liquid drugs injection, pharmaceutical composition consist of recombinant methionyl human G-CSF 100 weight parts, N.F,USP MANNITOL 5～10 weight parts; When described formulation is freeze-dried powder, pharmaceutical composition consist of recombinant methionyl human G-CSF 100 weight parts, N.F,USP MANNITOL 50～100 weight parts.
Below technical scheme of the present invention is made further explanation.
Employing gel chromatographic columns of the present invention is processed the fermenting culture of recombinant methionyl human G-CSF, by the meticulous control to chromatographic condition, makes the mass recovery of the hG-GSF that obtains by step 4 reach 48.9%, and specific activity is 4.4 * 10
8iU/mg, purity is 89.6%.Through step 5, carry out further purifying, its purity 99.75%, the specific activity that the rate of recovery of step 5 is 96.3%, hG-GSF is 4.3 * 10
8iU/mg.Its total mass recovery rate of preparation method of the present invention can reach 46.8%, and purity reaches 99.75%, and specific activity is 4.3 * 10
8iU/mg.
The invention still further relates to the pharmaceutical composition of recombinant methionyl human G-CSF, and this pharmaceutical composition can be prepared into multiple formulation.In this pharmaceutical composition, can add the additives such as appropriate vehicle, stablizer.In the present invention, selected the good N.F,USP MANNITOL of effect.Wherein the selection of excipient substance and preparation method can select ordinary method preparation.Specification can, according to the needs in market, be prepared into the different specifications such as 50 μ g/ prop up, 100 μ g/ prop up, 150 μ g/ prop up, 300 μ g/ prop up.
Preparation method of the present invention has overcome in prior art the low technical barrier of productive rate in recombinant methionyl human G-CSF biofermentation method preparation process, and is applicable to large-scale industrial production.
The specific embodiment of the present invention only limits to further explain and explanation the present invention, not to Composition of contents restriction of the present invention.
Embodiment
Embodiment 1 prepares recombinant methionyl human G-CSF:
1. select engineering bacteria DH5 α-PBV220-hGCSF bacterial strain to carry out fermentation culture;
2. by TE damping fluid washing 3 times for the thalline of collecting, then adopt ultrasonication 0.5～1 hour, low-temperature centrifugation, collects inclusion body; Add again inclusion body washings, under 4 ℃ of conditions, mix 30 minutes, 4 ℃ of low-temperature centrifugations; TE damping fluid is: 20mmol/LTris-HCl+1mmol/L EDTA, pH8.0; Inclusion body washings is: 20mmol/L Tris-HCl+1mmol/L EDTA+2mmol/L urea+0.5%Tritonx-100, pH8.0; The volume ratio of inclusion body weight and inclusion body washings is 1:10;
3. in inclusion body, add solubilization of inclusion bodies liquid, 4 ℃ of conditions are after lower 12 hours, the centrifugal inclusion body extracting solution that obtains; Described solubilization of inclusion bodies liquid is: 20mmol/L Tris-HCl+1mmol/L EDTA+7mmol/L Guanidinium hydrochloride+20mmol/L DTT, pH8.0; Inclusion body quality is 1:10 with the ratio of the volume of solubilization of inclusion bodies liquid;
4. adopt SephacrylS-200 gel column, under 4 ℃ of conditions, first use moving phase balance, extracting solution upper prop chromatographic condition is:
Moving phase is: 0.15mol/L NaCl+0.05mol/L Tris-HCl+1mmol/L EDTA+2mmol/L urea+0.8mmol/LGSH+0.2mmol/L GSSG, pH8.0;
Flow velocity is 2mL/min;
Check wavelength is 280nm;
Collect the recombinant methionyl human G-CSF cut of renaturation;
5. people's rhG-CSF cut of the renaturation of collection being adjusted to pH with 10% Glacial acetic acid is 4.2, low-temperature centrifugation 30 minutes supernatant liquor; It is the sodium-acetate balance of 4.2 20mmol/L that SephadexG25 post first adopts pH; SephadexG25 post on supernatant liquor, uses elutriant wash-out, and elutriant is: the sodium-acetate of sodium-chlor+15mmol/L of 10mmol/L, and pH is 4.4; Collect main peak and carry out separation, obtain recombinant human colony-stimulating factor.
After testing, the mass recovery 46.8% of preparation-obtained recombinant human colony-stimulating factor, purity (HPLC) is 99.76%, specific activity is 4.3 * 10
8iU/mg.The recombinant human colony-stimulating factor preparing shows band at 18.8kDa place through SDS-PAGE.
6. by the recombinant human colony-stimulating factor obtaining, with water for injection, dilute, according to 100 weight part recombinant human colony-stimulating factors, add the ratio of N.F,USP MANNITOL 5 weight parts, through 0.22 μ m millipore filter, carry out after Sterile Filtration, be prepared into the liquid drugs injection that 100 μ g/ prop up.Or be prepared into the different specifications such as 50 μ g/ prop up, 150 μ g/ prop up, 300 μ g/ prop up.
The impact of urea concentration on hG-GSF in experimental example 1:SephacrylS-200 gel column elutriant
In change embodiment 1, the concentration (other conditions and step are all identical with embodiment 1) of urea in elutriant, studies its impact on recombinant human colony-stimulating factor yield and specific activity, and its result is as shown in table 1:
Table 1:
| Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 | Comparative example 5 |
| Urea (mmol/L) | 1 | 1.5 | 1.75 | 2.25 | 2.5 |
| Mass recovery (%) | 40.1 | 40.4 | 45.2 | 43.7 | 37.8 |
| Specific activity (IU/mg) | 3.1×10 8 | 3.2×10 8 | 3.6×10 8 | 4.1×10 8 | 3.4×10 8 |
The impact of elutriant PH on hG-GSF in experimental example 2:SephacrylS-200 gel column
In change embodiment 1, in elutriant, other conditions of PH(are all identical with embodiment 1 with step), study its impact on recombinant human colony-stimulating factor yield and specific activity, its result is as shown in table 2:
Table 2:
| Comparative example 6 | Comparative example 7 | Comparative example 8 | Comparative example 9 | Comparative example 10 | |
| PH | 7 | 7.5 | 8 | 8.2 | 8.5 |
| Mass recovery (%) | 41.2 | 41.6 | 44.7 | 42.9 | 40．5 |
| Specific activity (IU/mg) | 2.1×10 8 | 2.5×10 8 | 3.9×10 8 | 3.5×10 8 | 3.2×10 8 |
The impact of GSH and GSSG mol ratio in experimental example 3:SephacrylS-200 gel column elutriant PH
In change embodiment 1, in elutriant, other conditions of PH(are all identical with embodiment 1 with step), study its impact on recombinant human colony-stimulating factor yield and specific activity: its result is as shown in table 3:
Table 3:
| Comparative example 11 | Comparative example 12 | Comparative example 13 | Comparative example 14 | Comparative example 15 | |
| The mol ratio of GSH:GSSG | 1：2 | 1：3 | 1：3.5 | 1：4.5 | 5 |
| Mass recovery (%) | 41.7 | 42.0 | 43.5 | 41.2 | 39.7 |
| Specific activity (IU/mg) | 2.2×10 8 | 2.7×10 8 | 3.8×10 8 | 4.1×10 8 | 3.0×10 8 |
Experimental example 4:
Other conditions of PH(and the step of the elutriant of change SephadexG25 post are all identical with embodiment 1), study its impact on recombinant human colony-stimulating factor purity and specific activity: its result is as shown in table 4:
Table 4:
| Comparative example 11 | Comparative example 12 | Comparative example 13 | Comparative example 14 | Comparative example 15 | |
| PH | 3.5 | 4 | 4.2 | 4.8 | 5 |
| Purity (%) | 97.45 | 99.32 | 99.53 | 99.47 | 98.2 |
| Specific activity (IU/mg) | 3.8×10 8 | 4.2×10 8 | 4.1×10 8 | 3.7×10 8 | 3.1×10 8 |
Experimental example 5: test of long duration
3 batches 101,102,103 of the recombinant human colony-stimulating factor injection liquid that the embodiment of the present invention 1 is prepared, simulation listing packing, carry out following stability test: put in sealing clean container, at 6 ℃ ± 2 ℃, under 60% ± 5%RH part, place 24 months, at duration of test respectively at the 3rd, 6,9,12,18,24 samplings at the end of month once, each stability high spot reviews project is tested.Test-results is as shown in table 5:
Table 5: long-term test results
From long-term test results, recombinant human colony-stimulating factor injection liquid of the present invention is investigated through test of long duration for 24 months, and considerable change does not all occur indices.The stability that confirms recombinant human colony-stimulating factor injection liquid of the present invention is good.
Claims (4)
1. a preparation method for recombinant methionyl human G-CSF, is characterized in that, described preparation method's step is:
(1) select the engineering bacteria of recombinant methionyl human G-CSF to carry out fermentation culture;
(2) fermention medium gained thalline is carried out to ultrasonication, collect inclusion body, with inclusion body washings, wash rear low-temperature centrifugation;
(3) in inclusion body, add solubilization of inclusion bodies liquid, under 4 ℃ of conditions after 8～12 hours, the centrifugal inclusion body extracting solution that obtains;
(4) under 2～8 ℃ of conditions, adopt SephacrylS-200 gel column to carry out separation to extracting solution, collect the recombinant methionyl human G-CSF cut of renaturation; Described chromatographic condition is: moving phase is: 0.15mol/L NaCl+0.05mol/LTris-HCl+1mmol/L EDTA+2mmol/L urea+0.8mmol/L GSH+0.2mmol/L GSSG, pH7.8; Flow velocity is 2mL/min; Check wavelength is 280nm;
(5) adopt SephadexG25 post to carry out separation: it is the sodium-acetate balance of 4.0～4.5 20mmol/L that described SephadexG25 post first adopts pH, it is 4.2 that people's rhG-CSF cut of the renaturation of collection is adjusted to pH with 10% Glacial acetic acid again; Low-temperature centrifugation obtains supernatant liquor in 20～40 minutes, and SephadexG25 post on supernatant liquor, uses elutriant wash-out, and described elutriant is: the sodium-acetate of sodium-chlor+15mmol/L of 10mmol/L, and pH is 4.4; Collect main peak and carry out separation, obtain recombinant methionyl human G-CSF.
2. the preparation method of recombinant methionyl human G-CSF according to claim 1, is characterized in that, in step (2), by TE damping fluid washing 1～3 time for the thalline of collecting, then adopt ultrasonication 0.5～1 hour, low-temperature centrifugation, collects inclusion body; Add again inclusion body washings, under 0～4 ℃ of condition, mix 20～60 minutes low-temperature centrifugation.
3. the preparation method of recombinant methionyl human G-CSF according to claim 1, is characterized in that, in step (4), described SephacrylS-200 gel column is first used moving phase balance, and then sample introduction carries out separation to inclusion body extracting solution.
4. the preparation method of recombinant methionyl human G-CSF according to claim 1, is characterized in that, in step (5), it is the sodium-acetate balance of 4.2 20mmol/L that described SephadexG25 post first adopts pH.
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Citations (2)
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| CN1814779A (en) * | 2005-12-20 | 2006-08-09 | 山东泉港药业有限公司 | Method for producing recombinant human granulocyte colony stimulating factor |
| CN101045742A (en) * | 2006-03-27 | 2007-10-03 | 华北制药集团新药研究开发有限责任公司 | Dipurification process of recombinant humangranulocyte colony stimulating factor |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1814779A (en) * | 2005-12-20 | 2006-08-09 | 山东泉港药业有限公司 | Method for producing recombinant human granulocyte colony stimulating factor |
| CN101045742A (en) * | 2006-03-27 | 2007-10-03 | 华北制药集团新药研究开发有限责任公司 | Dipurification process of recombinant humangranulocyte colony stimulating factor |
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| High Recovery Refolding of rhG-CSF from Escherichia coli,Using Urea Gradient Size Exclusion Chromatography;Chaozhan Wang,et al;《Biotechnology Progress》;20080108;第24卷(第1期);209-213 * |
| 凌明圣等.大肠杆菌表达的重组人粒细胞-巨噬细胞集落刺激因子的纯化.《生物工程学报》.1995,第11卷(第3期),217-221. |
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| 王骊丽 耿信笃.源于大肠杆菌蛋白的表达、液相色谱复性与纯化新进展.《中国科学》.2009,第39卷(第8期),711-727. |
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