CN1142281C - Preparation of recombined erythropoietin preparation - Google Patents

Abstract

The present invention discloses a method for vastly producing a human erythropoietin preparation, which comprises the steps: a nutrient culture medium added with insulin and transferrin is used for cultivating converted mammalian cells carried with human erythropoietin DNA encoding sequences under the specified culture conditions to obtain high expression of the protein product; and then the harvest liquid containing rich human erythropoietin is orderly processed by affinity chromatography, ion exchange, reversed phase high-efficiency liquid phase and gel filtration and purification by RP-HPLC and gel so as to obtain high-purity human erythropoietin having high biological activity.

Description

The preparation production method of recombinant human erythropoietin's preparation

Technical field

The present invention relates to utilize the eukaryotic cell expression system of carrying the proteinic nucleotide sequence of coding human erythropoietin, scale operation human erythropoietin method of protein.Particularly relate to the high yield is the separation of purpose and the method for purifying height ratio living person erythropoietin.

Background technology

Human erythropoietin (hEPO) is the glycoprotein of a kind of about 36KD of molecular weight that is mainly produced by kidney.This protein can act on bone marrow stem cell effectively as a kind of normal hormone, regulates and stimulation reticulocyte and erythrocytic generation and differentiation, thereby keeps the interior normoerythrocyte quantity of peripheral circulation.Therefore can be used for treating various forms of anaemias, particularly renal anemia clinically and produce the not enough red corpuscle deficiency disease that causes because of this hormone.

U.s. patent application serial number NO.570,075 (New youk, University) and United States Patent (USP) NO.4,677,195 (Genetics Institute, Inc.) disclose respectively with the method for anti-phase immunochromatographic method from natural origin purifying human erythropoietin, the latter is 160 with the erythropoietin protein matter specific activity of anti-phase immunochromatographic method preparation, the 000IU/280nm absorbance unit.Chinese patent CN 85106191A (Corriy-Ellge Corp) discloses and has utilized eukaryotic cell and the production of procaryotic cell expression system to have the method that the natural human promoting erythrocyte generates active protein (human erythropoietin).Wherein the hEPO product activity in vivo with escherichia coli expression that obtains with reversed phase high efficiency liquid phase method has only 120-720IU/mg protein, particularly mention in the document, the contriver is respectively 2589 IU/ml and 3089 IU/ml with eukaryotic cell expression system (Chinese hamster ovary cell) acquisition EPO product is external with activity in vivo.In addition, the purity of its expression product is about 95%, and clearly lacks enough molecule homogeneity.Activity as for its prepared human erythropoietin analogue is the 1/4-1/10 of its " parental generation ".European patent application 0,232 034 public use host people cell (Hala cell) is made the method for host's fermentative production rhEPO, and wherein having mentioned its lytic activity is 490 units.PCT patent publication No. W088/00241, the method of the Apa I restriction fragment of public use COS-7 (monkey kidney) and BHK (hamster kidney) cell expression system expressing human erythropoietin gene, though should side's document do not describe the separation and the purification process of expression product, but the ratio work of the secretory product of pointing out in the embodiment 7 is 700 units/ml, estimates at the proteinic specific activity of 7800 units/ml that is equivalent to natural EPO.At last, european patent application 0,236 059 discloses to use and has had neo ^xThe recombinant vectors of selectable marker gene (linear transduction vector) is expressed in mammalian cell, and with the method for affinity chromatography purifying EPO, detecting the proteinic output of proof erythropoietin through the external beam radiotherapy immunization is 100-1500 unit/10 ⁶Cell/sky.

In sum, these current materials only with regard to the isolation and purification means of laboratory level expression product, all can not obtain gratifying effect.Particularly arrive the industrially scalable high volume production process with laboratory study, the technical difference that has essence, explore and a large amount of experiment accumulation without creativeness over a long time, although in the laboratory, successfully obtained required product, but will be with high yield, the low-cost gene engineering product that obtains high specific acitivity, as human erythropoietin, it will be impossible really realizing the commercialization of purpose product and obtain due interests.

Up to now, set up and in production practice, successfully used many from natural origin or from separating the transformant cell of DNA recombinant technology preparation and the culture thereof and the method for the purpose product that purifying is required, these methods comprise saltout, ultrafiltration, electrophoresis (as polyacrylamide gel electrophoresis, isoelectrofocusing, electrocapillary phoresis etc.), column chromatography (as ion exchange chromatography, gel chromatography, high performance liquid chromatography, affinity chromatography etc.).Because required molecular weight of product size, charging property are different with molecule electric charge number, wetting ability and iso-electric point, changing method required when separating with purified product or method combination and their order also will be complete different.Therefore finding a cover to be fit to the fermentation culture conditions that the suitability for industrialized production scale is used to recombinant bacterial strain or cell strain, to improve the product expression efficiency, and improve and separate and the purify DNA recombinant products, for example confirming the method for the recombinant human erythropoietin (rhEPO) that its effect is very clear and definite at present in clinical application, is that a this area has problem to be solved.

Summary of the invention

Eukaryotic cell (Chinese hamster ovary celI) expression system that carry coding human erythropoietin proteinic nucleotide sequence of the present invention by adopting this laboratory to make up utilizes specific substratum and culture condition and continuous chromatography method to realize.Its technical scheme main points may further comprise the steps:

Get frozen production cell strain, 39 ℃ of water-baths, centrifugal under aseptic technique.Add an amount of DMEM substratum that contains 5% foetal calf serum after abandoning supernatant, and be inoculated in the Tissue Culture Flask 37 ℃ of cultivations in the carbonic acid gas incubator.After treating the full culturing bottle wall of cell well-grown and card, continue to go down to posterity (passing the three generations) cultivates.State with microscope observing cell is fusiformis at cell, when growth conditions is good, discards old substratum,, is collected in the inoculation bottle cell dissociation with Digestive system.The cell quantity that is used to inoculate is 2.5 * 10 ⁶Individual/ml.

The expression employing volume that contains the recombinant cell strain of human epo gene is the successive type cell reactor (hereinafter to be referred as the cell jar) of 5L, adds cellulose carrier sheet (Disc), PBS damping fluid (pH7.0), connects good pipeline, autoclaving 1.5 hours.Treat to insert main frame after the cell jar is cooled to room temperature, and connect four kinds of gases such as air, oxygen, carbonic acid gas, nitrogen.Proofreaied and correct behind the pH electrode and the DO. electrode after, discharge the PBS damping fluid in the cell jar, add the DMEM substratum that contains 5% foetal calf serum, cell inoculation liquid is slowly added in the cell jar again, regulate the stirring revolution and be lower than 50rpm/ minute, temperature is 37 ℃, feed four kinds of gases, control DO.50-80%, pH7.0 carries out adherent culture for some time.Improve revolution then by 80-100rpm/ minute, carried out the interior amplification cultivation of cell jar ten days.(but differentiation and propagation that blood serum medium effective stimulus cell is arranged).The DMEM substratum that will contain foetal calf serum is replaced by the synthetic medium of serum-free, with the cultural method of continuous perfusion substratum, uses the fermentation condition of AFS software control cell, comprises temperature, dissolved oxygen, pH value etc.The cultivation of results is based on 4-8 ℃ of low temperature storage.The expression output of hEPO can reach 10,000 IU/ml in the substratum that this working condition is gathered in the crops down.The synthetic medium of using serum-free simultaneously can reduce the content of heteroproteins in the purge process, increases the capacity of each chromatography chromatographic column, prolongs its work-ing life, effectively improves half-finished purity.The synthetic medium of used serum-free is the CHO-S-SFM-II substratum, and the amount of insulin of wherein adding is 1nM-10mM, Transferrins,iron complexes add-on 0.1nM-100nM, and the glucose add-on is the 1-10 grams per liter, the sodium bicarbonate add-on is the 2-4 grams per liter.

Get the substratum membrane filtration that obtains, with CM affinity column on certain flow velocity (in advance with the activation of NaCl-HAc-Virahol, 20mM Tris.HCl level pad balance), use level pad balance CM post more then.Adopt 0.2M NaCl-20mM Tris elutriant to carry out gradient elution, collect the activated elution peak dialysis tubing of packing into, dialyzed overnight in 10mM Tris dialyzate with certain flow rate.With the protein soln membrane filtration of the desalination of dialysing, with DEAE ion exchange column on certain flow velocity (using 10Mm Tris level pad balance in advance).Adopt the elutriant of 0.1M NaCl-Tris to carry out gradient elution, collect activated elution peak, (RP-HPLC chromatography column filler is C4 to last RP-HPLC, use acetonitrile-trifluoroacetic acid balance in advance) chromatography column, use 10% this chromatography column of acetonitrile solution balance then, the acetonitrile solution with 10-70% carries out gradient elution again.Collect activated elution peak, last gel chromatography column (using 20mM citrate buffer balance in advance) is used 20mM citrate buffer balance and wash-out again, collects activated elution peak (being hEPO).The ratio of sampling and measuring hEPO is lived, protein content, with the human serum albumin that adds 0.5% among the hEPO, makes the hEPO finished product preparation after the sterile filtration after quality inspection is qualified.Through the rh-EPO of this technology purifying, it can reach 2 * 10 than work ⁵IU/mg.

Embodiment

By embodiment the present invention is described for example further below, but these embodiment and not limiting the present invention in any way.

Embodiment one: the propagation of cell

Get the freeze-stored cell strain, 39 ℃ of water-baths, centrifugal under the aseptic technique (1000rpm * 5 minute).Abandon and add 5ml behind the supernatant and contain the DMEM substratum of 5% foetal calf serum (every 10L contains DMEM 135 grams, foetal calf serum 500ml, gentamicin 500,000 units, glucose 13.5 grams, sodium bicarbonate 24.5 grams) piping and druming evenly, be inoculated in the 100ml Tissue Culture Flask, add the DMEM substratum that 10ml contains 5% foetal calf serum again, in 37 ℃, the carbonic acid gas incubator (production of Forma company) of 5%CO2, cultivate.Treat that cell growth state is good, stick the culturing bottle wall after, import in four 500ml culturing bottles, and when cell is sticked bottle wall once more, import the culturing bottle of 32 bottles of 500ml into.Use the microscope observing cell growth conditions, when cell is in logarithmic phase, discard old substratum, with Digestive system (every liter contains EDTA 0.2g, glucose 1g, pancreatin 2g pH7.6) with cell dissociation, is collected in the inoculation bottle.Being used for inoculating cell quantity is 2.5 * 10 ⁶Individual/ml, be total to 900ml.

Embodiment two: the expression of erythropoietin

It is successive type cell reactor (the CELLIGEN PLUS of 5L that the cell jar adopts volume, NBS company makes), the PBS damping fluid (pH7.0) of adding 150 gram cellulose carrier sheets (Disc, LifeGen Co. produces), 3.5L, connect good pipeline, autoclaving is 1.5 hours under 121 ℃, 15 pounds.Treat to insert main frame after the cell jar is cooled to room temperature, connect four kinds of gases such as air, oxygen, carbonic acid gas and nitrogen, proofreaied and correct pH electrode and DO. electrode.Discharge the PBS damping fluid in the cell jar, adding 2500ml contains the DMEM substratum of 5% foetal calf serum, again 900ml cell inoculation liquid is slowly added in the cell jar, regulating revolution is 50rpm/ minute, feed four kinds of gases (oxygen, nitrogen, carbon dioxide gas and pressurized air), control DO.50%, pH7.0 makes cell attachment growth four hours.Improve revolution then by 80 rpm/ minutes, continued amplification cultivation ten days.Substratum is replaced with the synthetic medium of serum-free, and (every 10L contains CHO-S-SFM-II9L, gentamicin 500,000 units, Regular Insulin 1mM, Transferrins,iron complexes 20nM, glucose 25 grams, sodium bicarbonate 30.5 grams), cultural method with continuous perfusion substratum, use AFS software (design of NBS company is produced) control cell fermentation condition (37 ℃ of temperature, DO.40-80%, pH 6.5-7.5, substratum perfusion flow are 3L/ days), results liquid is in the storage down of 4-8 ℃ of low temperature.

Embodiment three: the purifying of hEPO

Get and obtain substratum 40L, use the 0.22um membrane filtration, what be splined on volume and be 2500ml uses 1.4MNaCl-40% Virahol-0.1M HAc 13000ml (pH 3.0) activation in advance, wash with the 10000ml tri-distilled water, use 20mM Tris (pH 7.0) 10000ml level pad equilibrated CM-Affi Blue Gel post (production of BioRad company) at last, last sample flow velocity is that the last sample of 30ml/min finishes, and uses level pad balance cylinder again, washes the foreign protein that can not adsorb off.The protein that adopts 0.2M NaCl-20mM Tris (pH 7.0) elutriant to carry out gradient elution absorption, elution speed 40ml/min.Collect elution peak 3000ml, protein content is 1mg/ml.With the protein solution of the collecting dialysis tubing of packing into,,, divide and change liquid four times by 15 times of volume calculation with 10mM Tris.HCl (pH 7.0) dialyzate dialyzed overnight.

With the protein soln of the desalination of dialysing 0.22um membrane filtration, being splined on volume is the Tris.HCl of 10mM in advance (pH 7.0) the 50000ml level pad equilibrated DEAE-Sepharose Fast Flow post (production of Parmacia company) of 2500ml, and last sample flow velocity is 4ml/min.Last sample finishes, and adopts the elutriant of 0.1M NaCl--Tris (pH 7.0) to carry out gradient elution, and elution flow rate is 40ml/min.Collect the 3rd elution peak 1800ml, protein content is 0.37mg/ml, makes the crude product that contains hEPO and prepare to be used for being further purified of RP-HPLC after sterile filtration.

Column packing is C4 (300A, 15um, Separation Group produce), column volume be RP-HPLC (Warters company produces, the 2000 types) chromatography column of 500ml with 10% acetonitrile solution (0.1% trifluoroacetic acid) balance, flow velocity is 60ml/min.Get sample on the 1800ml crude product, flow velocity is 15ml/min; Behind the end of the sample with 10% acetonitrile solution (containing 0.1% trifluoroacetic acid) balance C4 post.Acetonitrile solution with 10-70% carries out gradient elution again.Collect activated elution peak 330ml, protein content is 1.0mg/ml.

What gel-filtration was adopted is Sephades G-25 post (productions of Parmacia company), dress column volume 3000ml, and usefulness 20mM citrate buffer 6000ml balance is got sample on the sample behind the RP-HPLC purifying, and flow velocity is 1ml/min.Last sample finishes, and uses 20mM citrate buffer balance and wash-out again, collects elution peak 250ml, and protein content is 1.2mg/ml.The ratio that sampling is measured hEPO in the work in-process with microplate reader (Bio-Bad company produces, 3550 types) is lived, and measures protein concn with ultraviolet spectrophotometer (BECKMAN company produces, the DU-640 type); After quality inspection is qualified, add 0.5% human serum albumin and sterile filtration, make the hEPO finished product.Through the hEPO of this technological process of production purifying, it can reach 2 * 10 than work ⁵IU/mg.

Claims (2)

1. the method for an expressing human erythropoietin, comprise for the Chinese hamster ovary celI of the nucleotide sequence that contains the erythropoietin of encoding and cultivating, it is characterized in that, Chinese hamster ovary celI inoculation liquid amplification cultivation in the cell jar that will contain the nucleotide sequence of the erythropoietin of encoding earlier with the DMEM substratum that contains foetal calf serum, again substratum is replaced by the synthetic medium of serum-free, perfusion is cultivated continuously, contains Regular Insulin, Transferrins,iron complexes, sugar and sodium bicarbonate in the synthetic medium of described serum-free.

2. according to the process of claim 1 wherein that the synthetic medium of described serum-free is the CHO-S-SFM-II substratum, wherein contain Regular Insulin 1nM-10mM, Transferrins,iron complexes 0.1nM-100nM, glucose 1-10 grams per liter, sodium bicarbonate 2-4 grams per liter.