CN102040659A - Method for separating and purifying recombinant human erythropoietin - Google Patents
Method for separating and purifying recombinant human erythropoietin Download PDFInfo
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Abstract
The invention relates to the field of recombinant protein purification in gene engineering, in particular to a method for separating and purifying recombinant human erythropoietin by using the anion exchange chromatography. The method comprises the following steps of: (1) carrying out blue gum chromatography on a sample; (2) ultrafiltering and concentrating a product obtained from the blue gum chromatography; (3) carrying out first ion exchange chromatography on the an ultrafiltrated and concentrated product; (4) carrying out C4 reversed phase chromatography on a product obtained from the first ion exchange chromatography; (5) carrying out second ion exchange chromatography on a product obtained from the C4 reversed phase chromatography; and (6) carrying out S-200 molecular sieve chromatography on the product obtained from the second ion exchange chromatography to obtain recombinant human erythropoietin. The purity of rhEPO (Recombinant Human Erythropoietin) purified by the method of the invention can reach more than 99%, the content of sialic acid reaches more than 9.5 mol/mol EPO, and the biological specific activity in vivo reaches more than 1.4*10<5> IU/mg. The invention makes improvement on the basis of the traditional technological method, not only solves the problem of production scale, but also improves the quality of rhEPO products.
Description
Technical field
The present invention relates to genetically engineered recombinant protein purification field, relate in particular to a kind of method that adopts the anion-exchange chromatography technology recombinant human erythropoietin to be carried out separation and purification.
Background technology
(Erythropoietin EPO) is found in 1906 to erythropoietin the earliest, is a kind of glycoprotein, belongs to the sialoglycoprotein hormone, and protein portion is made up of 166 amino acid, and molecular weight is 34KD.Its encoding gene is a single copy gene, is positioned people's long-armed 21 districts of No. 7 karyomit(e)s.Difference according to its sugared type structure can be divided into two kinds of α, β, and the α type contains 34% carbohydrate, and the β type contains 26% carbohydrate, and two types all identical on effects such as biological characteristics, antigenicity.The physiological action of erythropoietin is to stimulate oxyphorase or erythrocytic formation in the marrow, is mainly generated and entered marrow by circulation blood by kidney to play a role.When people's renal function generation grievous injury, as under the situation of the acute and chronic renal failure or the nephrectomy, the generation of EPO reduces, and anaemia occurs.Therefore, EPO all has better curative effect to most renal anemia patients.
1985, the success of people EPO gene clone and expression becomes a reality the preparation of recombinant human erythropoietin, recombinant human erythropoietin (rhEPO) adopts reverse transcription PCR technology human cloning EPO-cDNA, be inserted in the eukaryotic expression vector, make up recombinant expression plasmid, and be transformed in the Chinese hamster ovary celI, filter out the engineering cell strain of high efficiency stable expression people EPO.Cultivate this project cell, the results supernatant carries out the rhEPO separation and purification of protein.RhEPO has structure and the physiological action identical with natural people's erythropoietin, it is the choice drug of treatment renal anemia, the red corpuscle that also can be used for the surgery peri-operation period is mobilized, the anaemia that treatment cancer and cancer radiation chemotherapy cause, the treatment acquired immune deficiency syndrome (AIDS) anemia etc. that occurs together, rhEPO is the most successful genetically engineered drug of human development so far.Along with further investigation, find that all there is effect preferably the aspects such as anaemia of rhEPO due to treatment gynaecology anaemia, pregnant and lying-in women's anaemia, rheumatoid arthritis to the rhEPO clinical application.
At present existing recombinant human erythropoietin separation purification method has following shortcoming: complex operation, each treatment capacity is not high, technology amplification difficulty is big, be not suitable for scale operation, and final rhEPO purity only can reach more than 95%, sialic acid content is more than the 9.0mol/molEPO, and the biology specific activity is 1.2 * 10 in the body
5IU/mg.
Need badly and set up a kind of suitable scale operation and low recombinant human erythropoietin (rhEPO) separation purification method of production cost, to satisfy a large amount of patients' demand.
Summary of the invention
For addressing the above problem, main purpose of the present invention is to provide a kind of process stabilizing, amplifies, is fit to scale operation and the low recombinant human erythropoietin separation purification method of production cost easily.
For achieving the above object, technical scheme of the present invention is:
A kind of recombinant human erythropoietin separation purification method may further comprise the steps:
1) sample carries out blue glue-line and analyses;
2) described blue glue-line division thing carries out ultrafiltration and concentration;
3) described ultrafiltration and concentration product carries out ion exchange chromatography I, 10mmol/L Tris-HCl damping fluid balance columns bed with pH6.5～7.5, use pH6.5～7.5 behind the last sample earlier, the 10mmol/L Tris-HCl buffer solution elution that contains 0.03～0.07mol/L NaCl is to baseline, use pH6.5～7.5 again, contain the 10mmol/L Tris-HCl buffer solution elution of 0.08～0.15mol/L NaCl;
4) described ion exchange chromatography I product carries out the C4 reversed phase chromatography;
5) the reverse chromatography product of described C4 carries out ion exchange chromatography II, 10mmol/L Tris-HCl damping fluid balance with pH6.5～7.5, behind the last sample earlier with pH3.7～4.7, contain the buffer solution elution post bed of 4～8mol/L urea, 0.5～2mmol/L glycine, be eluted to 10～15 column volumes of 10mmol/L Tris-HCl damping fluid drip washing of using pH6.5～7.5 behind the baseline again, at last with pH6.5～7.5, contain the 10mmol/L Tris-HCl buffer solution elution of 0.1～0.4mol/L NaCl;
6) described ion exchange chromatography II product carries out the S-200 sieve chromatography, obtains recombinant human erythropoietin albumen.
Sample is collected liquid for the perfusion culturing cell in the described step 1);
Described step 1) is specially: with pH6.5～7.5, contain the 20mmol/L Tris-HCl damping fluid balance Blue Sepharose 6 Fast Flow chromatography columns of 0.1～0.3mo l/L NaCl, behind the last sample, earlier with the drip washing of described damping fluid balance liquid to baseline, again with pH6.5～7.5, contain the 20mmol/L Tris-HCl buffer solution elution of 1.0～1.4mol/L NaCl, collect the rhEPO peak.
Preferably, step 1) is specially: with pH7.0, contain the 20mmol/LTris-HCl damping fluid balance Blue Sepharose 6 Fast Flow chromatography columns of 0.2mol/L NaCl, behind the last sample, earlier with the drip washing of described damping fluid balance liquid to baseline, again with pH7.0, contain the 20mmol/L Tris-HCl buffer solution elution of 1.3mol/L NaCl, collect the rhEPO peak.
Described step 2) be specially: the 10mmol/L Tris-HCl damping fluid with pH6.5～7.5 carries out ultrafiltration and concentration, is concentrated into specific conductivity and damping fluid specific conductivity and differs 100～200 μ S/cm.
Preferably, step 2) be specially: the 10mmol/L Tris-HCl damping fluid with pH7.0 carries out ultrafiltration and concentration, is concentrated into specific conductivity and damping fluid specific conductivity and differs 180 μ S/cm.
Described step 3) is specially: with the 10mmol/L Tris-HCl damping fluid balance columns bed of pH6.5～7.5, behind the last sample earlier with pH6.5～7.5, contain the 10mmol/L Tris-HCl buffer solution elution of 0.03～0.07mol/L NaCl to baseline, again with pH6.5～7.5, contain the 10mmol/L Tris-HCl buffer solution elution of 0.08～0.15mol/L NaCl, collect DEAE I elution peak.
Preferably, step 3) is specially: with the 10mmol/L Tris-HCl damping fluid balance columns bed of pH7.0, behind the last sample earlier with pH7.0, contain the 10mmol/L Tris-HCl buffer solution elution of 0.04mol/L NaCl to baseline, again with pH7.0, contain the 10mmol/LTris-HCl buffer solution elution of 0.13mol/L NaCl, collect DEAE I elution peak.
Described step 4) is specially: with pH6.5～7.5, contain 4%～10% alcoholic acid 10mmol/LTris-HCl damping fluid balance C4 post, behind the last sample, earlier with pH6.5～7.5, contain 4%～10% alcoholic acid 10mmol/L Tris-HCl damping fluid drip washing to baseline, again with pH6.5～7.5, contain 40%～70% alcoholic acid 10mmol/L Tris-HCl buffer solution elution, collect elution peak.
Preferably, step 4) is specially: with pH7.0, contain 8% alcoholic acid 10mmol/LTris-HCl damping fluid balance C4 post, behind the last sample, earlier with pH7.0, contain 8% alcoholic acid 10mmol/L Tris-HCl damping fluid drip washing to baseline, again with pH7.0, contain 50% alcoholic acid 10mmol/L Tris-HCl buffer solution elution, collect elution peak.
Described step 5) is specially: with the 10mmol/L Tris-HCl damping fluid balance columns bed of pH6.5～7.5, behind the last sample earlier with pH3.7～4.7, contain the buffer solution elution post bed of 4～8mol/L urea, 0.5～2mmol/L glycine, be eluted to 10～15 column volumes of 10mmol/LTris-HCl damping fluid drip washing of using pH6.5～7.5 behind the baseline again, at last with pH6.5～7.5, contain the 10mmol/L Tris-HCl buffer solution elution of 0.1～0.4mol/L NaCl, collect DEAE II elution peak.
Preferably, step 5) is specially: with the 10mmol/L Tris-HCl damping fluid balance columns bed of pH7.0, behind the last sample earlier with pH4.0, contain the buffer solution elution post bed of 5mol/L urea, 1.5mmol/L glycine, be eluted to 15 column volumes of 10mmol/L Tris-HCl damping fluid drip washing of using pH7.0 behind the baseline again, at last with pH7.0, contain the 10mmol/L Tris-HCl buffer solution elution of 0.3mol/L NaCl, collect DEAE II elution peak;
Described step 6) is specially: with pH6.0～8.0, contain 20mmol/L Citric Acid-sodium citrate buffer balance S-200 post of 50～150mmol/L NaCl, last sample and wash-out, filter and aseptic collection with 0.22 μ m strainer simultaneously, collect the peak and be the recombinant human erythropoietin.
Preferably, step 6) is specially: with pH7.0, contain 20mmol/L Citric Acid-sodium citrate buffer balance S-200 post of 120mmol/L NaCl, last sample and wash-out filter and aseptic collection with 0.22 μ m strainer simultaneously, collect the peak and are the recombinant human erythropoietin.
Adopt twice anion-exchange chromatography among the present invention, the use of anion-exchange chromatography post (DEAE-Sepharose Fast Flow chromatography) for the second time, not only can carry out in various degree amplification according to producing needs, solve the industrial scale problem, and remove alcoholic acid simultaneously to rhEPO purity and body in the biology specific activity play the effect of further raising, this goes on foot the rhEPO removal that chromatography can be low with sialic acid content, improves the high proteic ratio of rhEPO of sialic acid content in the product.Adopt recombinant human erythropoietin separation purification method of the present invention, the rhEPO purity that purifying obtains can reach more than 99%, and sialic acid content reaches more than the 9.5mol/molEPO, and the biology specific activity reaches 1.4 * 10 in the body
5More than the IU/mg.The present invention improves on existing processing method basis, has not only solved the industrial scale problem, and has improved the rhEPO quality product.
Description of drawings
Fig. 1 is a recombinant human erythropoietin separation purification method process flow sheet of the present invention.
Embodiment
Below in conjunction with Figure of description recombinant human erythropoietin separation purification method of the present invention is described in further details.
Material is prepared: cultivate the serum-free medium that the recombined engineering cell strain that contains people's erythropoietin gene obtains to contain recombinant human erythropoietin (rhEPO) by the continuous perfusion of cell cultures jar.
The concrete steps of separation and purification rhEPO from above-mentioned nutrient solution:
1. blue glue-line is analysed (Blue Sepharose 6 Fast Flow chromatographies)
1.1 balance:, be 7.0 until effluent liquid pH with 0.2mol/L NaCl-20mmol/L Tris-HCl (pH7.0) damping fluid balance Blue Sepharose 6 Fast Flow chromatography columns.
1.2 last sample: with the 40ml/min flow velocity, with sample on the cell harvesting liquid after filtering.
1.3 drip washing: with 0.2mol/L NaCl-20mmol/L Tris-HCl (pH7.0) damping fluid with the flow velocity drip washing of 40ml/min to baseline.
1.4 wash-out: with 1.3mol/L NaCl-20mmol/L Tris-HCl (pH7.0) damping fluid with the flow velocity wash-out of 40ml/min and collect the EPO peak.
2. ultrafiltration and concentration
With blue glue elutriant 10mmol/L Tris-HCl, the pH7.0 damping fluid carries out ultrafiltration and concentration, differs about 180 μ S/cm up to concentrated solution specific conductivity and damping fluid specific conductivity.
3. ion exchange chromatography I (DEAE-Sepharose Fast Flow chromatography)
3.1 balance:, be 7.0 until effluent liquid pH with the flow velocity of 50mi/min 10mmol/L Tris-HCl (pH7.0) damping fluid balance columns bed.
3.2 last sample: the concentrated solution that ultrafiltration is obtained with the flow velocity of 40ml/min is splined on DEAE-Sepharose Fast Flow chromatography column.
3.3 drip washing:, drop to baseline until peak to be detected with of the flow velocity drip washing of 0.04mol/L NaCl-10mmol/L Tris-HCl (pH7.0) damping fluid with 40ml/min.
3.4 wash-out: with the flow velocity wash-out of 0.13mol/L NaCl-10mmol/LTris-HCl (pH7.0) damping fluid with 40ml/min, wash-out is collected liquid for DEAE I collects liquid, is collected in the serum bottle.
4.C4 reversed phase chromatography
4.1 balance: with 8% ethanol/10mmol/LTris-HCl (pH7.0) damping fluid balance VydacC4 post, equilibrium velocity is 40ml/min, 5 times of column volumes of drip washing.
4.2 last sample: with the flow velocity of 40ml/min DEAE I is collected liquid and be splined on Vydac C4 post.
4.3 drip washing: after going up sample and finishing, with 8% ethanol/10mmol/L Tris-HCl (pH7.0) damping fluid drip washing to baseline.
4.4 wash-out: with the flow velocity wash-out of 50% ethanol/10mmol/L Tris-HCl (pH7.0) damping fluid, when elution peak occurs, begin to be collected into till the elution peak end, be collected in the 5L serum bottle with 40ml/min.
5. ion exchange chromatography II (DEAE-Sepharose Fast Flow chromatography)
5.1 balance: with 10mmol/L Tris-HCl (pH7.0) damping fluid with the flow velocity balance of 40ml/min to effluent liquid pH be 7.0.
5.2 last sample: with the flow velocity of 40ml/min the C4 reversed phase chromatography is collected liquid and be splined on DEAE-Sepharose Fast Flow chromatography column.
5.3 drip washing: earlier be reduced to baseline with flow velocity drip washing to the ultraviolet detection value of 40ml/min, use 10mmol/LTris-HCl (pH7.0) damping fluid 15 column volumes of flow velocity drip washing again with 40ml/min with the damping fluid of 5mol/L urea/1.5mmol/L glycine (pH4.0).
5.4 wash-out: with containing the flow velocity wash-out of the damping fluid of 0.3mol/L NaCl/10mmol/L Tris-HCl (pH7.0) with 40ml/min, when rising, elution peak ultraviolet detection value begins to collect, finish to collect to being reduced to baseline, collect DEAE II elutriant in the 500ml saline bottle.This collects the liquid sialic acid content should be not less than 9.5mol/molEPO.
6.S-200 sieve chromatography
6.1 balance: with 20mmol/L Citric Acid-Sodium Citrate-120mmol/L NaCl (pH7.0) damping fluid 2 times of column volumes of flow velocity balance with 12ml/min.
6.2 last sample: going up the sample flow velocity is 12ml/min, and applied sample amount should be no more than 1/10 of column volume.
6.3 wash-out: with the flow velocity wash-out of 20mmol/L Citric Acid-Sodium Citrate-120mmol/L NaCl (pH7.0) damping fluid with 12ml/min.Begin to occur being collected into to stop when detected peaks drops to baseline collecting from elution peak.Use disposable filter, sterile filtration EPO product is recombinant human erythropoietin (rhEPO) the product stoste of final purifying to the Sterile Saline bottle.
Recombinant human erythropoietin (rhEPO) product after the separation and purification is through SDS-PAGE and HPLC check and analysis, and purity reaches more than 99.0%, reaches 1.5 * 10 with the external biological specific activity in the body
5More than the IU/mg, sialic acid content is not less than 9.5mol/mol EPO.Other test item all meets Chinese Pharmacopoeia current edition regulation.
The isolation and purification method of Recombinant Human Erythropoietin of the present invention (rhEPO) has the following advantages:
1) the purge process medium is big to the carrying capacity of albumen, and flow velocity is fast, and the operating time is short; Not only can process in a large number sample, also can carry out in various degree amplification according to need of production, solve the production scale problem.
2) purge process good reproducibility, and repeated regeneration utilizes process simple.
3) purge process is easy and simple to handle, does not need special reagent.
4) the rhEPO purity height that is obtained by the method by bringing up to more than 95% more than 99.0% of existing handicraft product purity, improves more than 4.0%.
5) the rhEPO activity yield height that is obtained by the method is increased to 15% by 10% of existing technology, has improved 50%.
6) by being increased to more than the 9.5mol/mol EPO about the 9.0mol/molEPO of sialic acid content by existing technology among the rhEPO of the method acquisition, improved more than 5%.
7) the biology specific activity is by 1.2 * 10 in the rhEPO body that is obtained by the method5IU/mg brings up to 1.4 * 105More than the IU/mg, improve about more than 17%.
Claims (8)
1. recombinant human erythropoietin separation purification method may further comprise the steps:
1) sample being carried out blue glue-line analyses;
2) described blue glue-line division thing is carried out ultrafiltration and concentration;
3) described ultrafiltration and concentration product is carried out ion exchange chromatography I, 10mmol/L Tris-HCl damping fluid balance columns bed with pH6.5～7.5, behind the last sample earlier with pH6.5～7.5, contain the 10mmol/L Tris-HCl buffer solution elution of 0.03～0.07mol/L NaCl to baseline, use pH6.5～7.5 again, contain the 10mmol/L Tris-HCl buffer solution elution of 0.08～0.15mol/L NaCl;
4) described ion exchange chromatography I product is carried out the C4 reversed phase chromatography;
5) the reverse chromatography product of described C4 is carried out ion exchange chromatography II, 10mmol/L Tris-HCl damping fluid balance columns bed with pH6.5～7.5, behind the last sample earlier with pH3.7～4.7, contain the buffer solution elution post bed of 4～8mol/L urea, 0.5～2mmol/L glycine, be eluted to 10～15 column volumes of 10mmol/L Tris-HCl damping fluid drip washing of using pH6.5～7.5 behind the baseline again, at last with pH6.5～7.5, contain the 10mmol/L Tris-HCl buffer solution elution of 0.1～0.4mol/L NaCl;
6) described ion exchange chromatography II product is carried out the S-200 sieve chromatography, obtain recombinant human erythropoietin albumen.
2. recombinant human erythropoietin separation purification method according to claim 1 is characterized in that, the described sample in the step 1) is collected liquid for the perfusion culturing cell.
3. recombinant human erythropoietin separation purification method according to claim 1, it is characterized in that, step 1) is specially: with pH6.5～7.5, contain the 20mmol/LTris-HCl damping fluid balance Blue Sepharose 6 Fast Flow chromatography columns of 0.1～0.3mol/L NaCl, behind the last sample, earlier with the drip washing of described damping fluid balance liquid to baseline, again with pH6.5～7.5, contain the 20mmol/L Tris-HCl buffer solution elution of 1.0～1.4mol/LNaCl, collect the rhEPO peak.
4. recombinant human erythropoietin separation purification method according to claim 1, it is characterized in that, step 2) be specially: the 10mmol/L Tris-HCl damping fluid with pH6.5～7.5 carries out ultrafiltration and concentration, is concentrated into concentrated solution specific conductivity and damping fluid specific conductivity and differs 100～200 μ S/cm.
5. recombinant human erythropoietin separation purification method according to claim 1, it is characterized in that, step 3) is specially, 10mmol/L Tris-HCl damping fluid balance columns bed with pH6.5～7.5, behind the last sample earlier with pH6.5～7.5, contain the 10mmol/LTris-HCl buffer solution elution of 0.03～0.07mol/L NaCl to baseline, use pH6.5～7.5 again, contain the 10mmol/L Tris-HCl buffer solution elution of 0.08～0.15mol/LNaCl, collect DEAE I elution peak.
6. recombinant human erythropoietin separation purification method according to claim 1, it is characterized in that, step 4) is specially: with pH6.5～7.5, contain 4%～10% alcoholic acid 10mmol/LTris-HCl damping fluid balance C4 post, behind the last sample, earlier with pH6.5～7.5, contain 4%～10% alcoholic acid 10mmol/LTris-HCl damping fluid drip washing to baseline, again with pH6.5～7.5, contain 40%～70% alcoholic acid 10mmol/L Tris-HCl buffer solution elution, collect elution peak.
7. recombinant human erythropoietin separation purification method according to claim 1 is characterized in that step 5) is specially:
10mmol/L Tris-HCl damping fluid balance columns bed with pH6.5～7.5, behind the last sample earlier with pH3.7～4.7, contain the buffer solution elution post bed of 4～8mol/L urea, 0.5～2mmol/L glycine, be eluted to 10～15 column volumes of 10mmol/L Tris-HCl damping fluid drip washing of using pH6.5～7.5 behind the baseline again, at last with pH6.5～7.5, contain the 10mmol/L Tris-HCl buffer solution elution of 0.1～0.4mol/L NaCl, collect DEAE II elution peak.
8. recombinant human erythropoietin separation purification method according to claim 1, it is characterized in that, described step 6) is specially: with pH6.0～8.0, contain 20mmol/L Citric Acid-sodium citrate buffer balance S-200 post of 50～150mmol/L NaCl, last sample and wash-out, filter and aseptic collection with 0.22 μ m strainer simultaneously, collect the peak and be the recombinant human erythropoietin.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112940101A (en) * | 2019-12-11 | 2021-06-11 | 深圳赛保尔生物药业有限公司 | Purification method of recombinant human erythropoietin |
| CN115032317A (en) * | 2022-06-29 | 2022-09-09 | 科兴生物制药股份有限公司 | Detection method of recombinant human erythropoietin |
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2009
- 2009-10-19 CN CN2009101105936A patent/CN102040659A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112940101A (en) * | 2019-12-11 | 2021-06-11 | 深圳赛保尔生物药业有限公司 | Purification method of recombinant human erythropoietin |
| CN115032317A (en) * | 2022-06-29 | 2022-09-09 | 科兴生物制药股份有限公司 | Detection method of recombinant human erythropoietin |
| CN115032317B (en) * | 2022-06-29 | 2024-03-26 | 科兴生物制药股份有限公司 | Method for detecting recombinant human erythropoietin |
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