CN102994547B - Recombinant human erythropoietin-CTP fusion protein production process and application - Google Patents
Recombinant human erythropoietin-CTP fusion protein production process and application Download PDFInfo
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Abstract
The invention discloses a recombinant human erythropoietin-CTP fusion protein production process. The process is characterized in that a microcarrier suspension perfusion culture technology or a serum-free suspension fed-batch culture technology is adopted as a recombinant engineering cell line culture manner. The invention also discloses a recombinant human erythropoietin-CTP fusion protein purifying process. Through optimization, a simple process flow is obtained, such that high-purity and high-activity recombinant human erythropoietin-CTP fusion protein is obtained. The invention also relates to the ticokinetics study of the recombinant human erythropoietin-CTP fusion protein in monkey bodies, and the application of the recombinant human erythropoietin-CTP fusion protein in treating rat renal anemia.
Description
Technical field
The invention belongs to bio-pharmaceuticals technology field.It is specifically related to a kind of producing and manufacturing technique of the fusion rotein of a kind of Recombinant Human Erythropoietin and human chorion gonadotrophic hormone beta subunit C-terminal peptide, comprises the acquisition of the recombined engineering cell strain of this fusion rotein, cell cultures and protein purification processing method and Recombinant Human Erythropoietin-CTP fusion rotein in the generation experiment of monkey medicine with to the application in Renal Anemia in Rats treatment.
Background technology
EPO is a kind of glycoprotein produced primarily of kidney, and molecular weight is about 35KD, is the major hormone regulating erythroid hematopoiesis in human body, in the propagation of CFU-E, differentiation and maturation, play important regulating and controlling effect.Current China is to EPO demand cumulative year after year, but to there is Half-life in vivo short for common EPO, the problems such as patient's administration number of times is frequent.Therefore how to extend its Half-life in vivo, to reduce dosage rate, become the study hotspot of current EPO medicine.Technique means conventional at present has three kinds, one is make EPO carry out PEGization (patent No. 200880021159.4), two is increase the glycosylation site (Aranesp of Amgen of the U.S. by amino acid mutation, go on the market), three is make itself and some high molecular weight proteins or high saccharification fragment be connected to form fusion rotein, existing bibliographical information has EPO-FC (see number of patent application 200880005733.7) at present, EPO-HAS (see number of patent application 200410053319.7) and EPO-CTP bibliographical information (" Development of a Long-ActingErythropoietin by Fusing the Carboxyl-Terminal Peptide of Human ChorionicGonadotropin_-Subunit to the Coding Sequence of Human Erythropoietin ") etc.
Fusion rotein of the present invention, with reference to above-mentioned patent and document basic design, increases at the C-terminal of EPO the peptide chain that a length of tape has human chorion gonadotrophic hormone beta subunit.Human chorionic gonadotrophin (hCG) is the glycoprotein hormones that a class of embryonic trophoblasts emiocytosis has important physiological function, connected to form with non covalent bond by two different subunit α, β, CTP small peptide is human chorion gonadotrophic hormone beta subunit C-terminal peptide.Human chorion gonadotrophic hormone beta subunit C-terminal peptide can increase sialic acid content, increase molecular weight, prolong half-life and do not affect epo protein activity, existing bibliographical information (" Development of aLong-Acting Erythropoietin by Fusing the Carboxyl-Terminal Peptide of HumanChorionic Gonadotropin_-Subunit to the Coding Sequence of HumanErythropoietin ") supports that it can make EPO Increased Plasma Half-life 2-3 doubly.
Because EPO is high glycated protein, 40% of its molecular weight is sugar, and after merging CTP small peptide, its degree of glycosylation is higher, and therefore the production of EPO-CTP fusion rotein also must select saccharification to modify more perfect mammalian cell expression system.The current more ripe production expression system of production of recombinant glycoprotein class medicine is mammalian cell expression system, and the most frequently used cell is Chinese hamster ovary cell (CHO).Current mammalian cell large-scale cultivation method can be divided into: adherent culture, suspension culture and Immobilized culture.Adherent culture refers to that cell attachment carries out monolayer culture at certain solid phase surface.Because cell is fixed on surface, do not need complicated cell cut-off equipment, be convenient to adopt perfusion culture.Suspension culture is the process of phalangeal cell free suspension growth in the reactor.Suspension culture system is mainly applicable to non-adherent dependent cell and cultivates.Immobilized culture is combined with water insoluble carrier by zooblast, then carry out a kind of method of cultivating.
In the present invention, the production of EPO-CTP fusion rotein can adopt two kinds of cell cultures modes, and namely microcarrier attaching perfusion culture and suspension flow add training method.Be characterized in adopting shearing force little, pass oxygen effective, hybrid mode is the rip current type bioreactor of rock type mixing.The first training method adopts microcarrier to attach suspension perfusion culture, microcarrier can sustenticular cell high-density, grow for a long time, and microcarrier suspension is in nutrient solution, higher mass transfer oxygen transfer efficiency can be realized, this kind of training method constantly can be supplemented fresh medium, discharges metabolic waste, be extended the cell expressing time, and utilize suspension culture nutritive substance, feature that dissolved oxygen mixes, thus improve recombinant protein output, the method for the high saccharification recombinant protein of the production for a kind of novel practical.The second training method is suspension feeding culture, is mainly used for being tamed by suspension and adapting to the cell of suspension culture.It is simple to operate, reliably flexible that suspension flow adds culture process, and particularly utilize rip current type bioreactor to pass oxygen effective, one-time reaction bag is easy to operate, greatly can shorten culture cycle, be easy to amplification culture scale, by the simple control of technique, EPO-CTP Production requirement can be met.
The present invention is studied the fusion rotein application in animal body utilizing this kind of production method to produce, and the medicine of fusion rotein in cynomolgus monkey demonstrates the transformation period being better than common EPO for experimental data.In Renal Anemia in Rats treatment, fusion rotein can reach one week long-acting be administered once.
Summary of the invention
Problem to be solved by this invention is mainly to optimize the producing and manufacturing technique defining a kind of Recombinant Human Erythropoietin-CTP fusion rotein, comprise the structure of the recombined engineering cell strain of Restruction people erythropoietin-CTP fusion rotein, and the separation and purification of protein method adopting carrier to attach perfusion culture technique or suspension feeding method and optimization obtains high reactivity, high purity fusion rotein, the experiment proved that this fusion rotein can reach the effect reducing administration frequency over the course for the treatment of.
The invention discloses nucleotide sequence and the aminoacid sequence of the fusion rotein of a kind of Recombinant Human Erythropoietin and human chorion gonadotrophic hormone beta subunit C-terminal peptide.
SEQ ID NO 1 is EPO-CTP nucleotide sequence
CCG
CCACC
GGGGTGCACGAATGTCCTGCCTGGCTGTGGCTTCTCCTGTCCCTGCTGTCGCTCCCTCTGGGCCTCCCAGTCCTGGGCGCCCCACCACGCCTCATCTGTGACAGCCGAGTCCTGGAGAGGTACCTCTTGGAGGCCAAGGAGGCCGAGAATATCACGACGGGCTGTGCTGAACACTGCAGCTTGAATGAGAATATCACTGTCCCAGACACCAAAGTTAATTTCTATGCCTGGAAGAGGATGGAGGTCGGGCAGCAGGCCGTAGAAGTCTGGCAGGGCCTGGCCCTGCTGTCGGAAGCTGTCCTGCGGGGCCAGGCCCTGTTGGTCAACTCTTCCCAGCCGTGGGAGCCCCTGCAGCTGCATGTGGATAAAGCCGTCAGTGGCCTTCGCAGCCTCACCACTCTGCTTCGGGCTCTGGGAGCCCAGAAGGAAGCCATCTCCCCTCCAGATGCGGCCTCAGCTGCTCCACTCCGAACAATCACTGCTGACACTTTCCGCAAACTCTTCCGAGTCTACTCCAATTTCCTCCGGGGAAAGCTGAAGCTGTACACAGGGGAGGCCTGCAGGACAGGGGACAGATCTTCCTCTTCAAAGGCCCCTCCACCTAGCCTTCCATCTCCATCTCGACTCCCTGGGCCTTCTGACACCCCTATCCTCCCACAA
AAAAGGAAAA
(square frame is initiator codon and terminator codon; Shade italic is EcoR I and Not I restriction enzyme site)
SEQ ID NO 2 is EPO-CTP aminoacid sequences
MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDRSSSSKAPPPSLPSPSRLPGPSDTPILPQ
SEQ ID NO 3 is EPO aminoacid sequences
MGVHECPAWLWLLLSLLSLPLGLPVLGAPPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLRGKLKLYTGEACRTGDR
SEQ ID NO 4 is CTP aminoacid sequences
SSSSKAPPPSLPSPSRLPGPSDTPILPQ
In fusion rotein of the present invention, CTP is human chorion gonadotrophic hormone beta subunit C-terminal 28 peptide, and position is at the C-terminal of fusion rotein.
The invention provides fusion rotein and carry out that zooblast is extensive, high-density, carrier attach perfusion culture technique or suspension feeding method, its key step comprises:
(1) cell inoculation step;
(2) the Growth of Cells stage;
(3) the cells produce stage;
The invention provides the purifying process after zooblast large scale culturing, concrete technology comprises:
(1) pre-treatment;
(2) affinity chromatography;
(3) ion-exchange;
(4) gel-filtration;
The fusion rotein that the present invention obtains, prove through treatment renal anemia rat experiment, this fusion rotein has the biological effect the same with EPO, and can reach the effect be administered once for a week.
Present invention also offers EPO-CTP cynomolgus monkey medicine for experimental data, prove that the transformation period of fusion rotein is better than common EPO, the mean terminal transformation period is 12.65h, and in commercially available import medicine profit blood treasured (common EPO) specification sheets, the transformation period is 5.9-7.5h.Clearance rate EPO-CTP 0.01ml/h/kg is obviously better than the precious 8.62ml/h/kg of sharp blood, illustrates EPO-CTP in vivo removing speed be slower than commercially available sharp blood treasured.Average retention time (MRT) EPO-CTP 70.77h, the precious 8.18h of sharp blood, can find out that EPO-CTP has certain long-acting.
Accompanying drawing explanation
Fig. 1 is the qualification of pMD18-T-EPO-CTP plasmid enzyme restriction
Fig. 2 is EPO-CTP fusion rotein structure
Fig. 3 is that EPO-CTP expression plasmid enzyme cuts qualification
Fig. 4 is the EPO-CTP fusion rotein electrophoresis of purifying
Fig. 5 is EPO-CTP fusion protein immunization engram analysis
Fig. 6 is EPO-CTP fusion rotein average blood serum concentration-time curve
Fig. 7 is that EPO-CTP fusion rotein treatment rat drug induced injury causes pharmacodynamics test in renal anemia body
Fig. 8 is that EPO-CTP fusion rotein treatment kidney of rats excision damage causes pharmacodynamics test in renal anemia body
Specific examples
Embodiment 1 expression strain construction process
To the EPO-CTP antigen-4 fusion protein gene on pMD18-T carrier be connected to, utilize Not I and EcoR I double digestion qualification positive colony (Fig. 1), order-checking.After obtaining the positive colony of correct sequence, be connected to by fusion gene on pcDNA carrier for expression of eukaryon, the expression strain built can carry out transfection CHO cell after extracting plasmid.EPO-CTP fusion rotein structural representation is shown in Fig. 2.
Get the plasmid of EPO-CTP fusion gene, Not I and EcoR I is used to carry out double digestion (Fig. 3), reclaim small segment, get pcDNA carrier for expression of eukaryon simultaneously, use Not I and EcoR I to carry out double digestion and reclaim large fragment, utilize T4 ligase enzyme to connect two fragments reclaimed, be transformed in bacillus coli DH 5 alpha, screening positive recombinant.By its called after pcDNA-EPO-CTP after double digestion qualification and sequence analysis are determined.This plasmid can carry out the transfection of Chinese hamster ovary celI.
Embodiment 2EPO-CTP fusion rotein transfection CHOK1 clone
Recombinant expression plasmid is proceeded to mammalian host cell line, to express EPO-CTP fusion rotein.In order to high-caliber stably express, preferred host cell system is CHOK1.The present invention adopts electricity to transform, and the linearizing DNA of 150 μ g adds 5 μ l salmon sperm dnas, CHOK1 host cell 5 × 106, and after mixing, 180V electric shock twice, turns electricity in the plate that rear cell suspension spreads with substratum.Treat to occur clone bunch, picking mono-clonal enlarged culturing in plate, through 3 time cloning screenings, obtain relatively stable overexpression cell line.
Embodiment 3 zooblast is extensive, high-density, carrier attach perfusion culture
Zooblast microcarrier attaches perfusion culture method, mainly comprises the inoculation of cell, initial growth phase, changes culture condition, subsequent growth stage and monitoring culture condition, and concrete steps are as follows:
1, the inoculation of cell.
Recover in cell bank one and express EPO-CTP cell strain, add 10% foetal calf serum with DMEM substratum and go down to posterity in T25 square vase three times, adjustment cell state.When cell is in logarithmic phase, with tryptic digestion, increase serum stops digestion, and the DMEM substratum adding 10% foetal calf serum carries out kind of a subchain amplification, and cell amplification goes down to posterity step by step according to the order of T25-T75-T150 square vase-2L rolling bottle.The bio-reactor with microcarrier is inoculated with the cell of rolling bottle digestion.After inoculation, cell density is 3 × 105 cells/ml,
2, initial growth phase.
After cell access production reactor, set every culture parameters, temperature 37 DEG C, PH6.9-7.2, DO40-70%, rotating speed 40-70 turn/min, and every day, sterile sampling, detected glucose and lactic acid content.
3, culture condition is changed
When glucose content is serum-free medium lower than changing nutrient solution during 1g/L, enter the expression phase, and carry out filling type cultivation, groundwater increment is for keeping glucose content about 2g/L.
4, downstream manufacturing stages
When culture cycle reaches 30-40 days, or glucose utilization 24 hours is lower than 0.5g/L, can stop cultivating.
5, culture condition is monitored
The index detected is needed to have temperature, pH, glucose concn, lactate concentration, ammonium salt concentration, glutamine concentration, osmolarity, express polypeptide or protein concn in culturing process.
Embodiment 4 purifying process
The present invention studies widely by going deep into, by the analysis of the physico-chemical property to EPO-CTP albumen, and groping of purifying process condition, thus the efficiently purifying technique achieving EPO-CTP.Because the expression of EPO-CTP of the present invention is present in fermentation supernatant, so fermented liquid obtains fermentation supernatant by centrifugal, filtration.The invention has the advantages that and determine preferably operational path, purifying process flow process is simple; The EPO-CTP fusion rotein of high-purity high-activity can be obtained, often liter of fermentor cultivation liquid can obtain at least 15mg, sialic acid is greater than 16mol/mol, specific activity is greater than 1.6 × 10
5the EPO-CTP fusion rotein that IU/mg, purity are greater than 95%.Purifying process concrete steps are as follows:
1, purifying pre-treatment
Get cell cultures good harvest liquid 30L, first 4500rpm, 15min, 4 DEG C centrifugal, then adopts 0.22 μm of form filter element filtering, filter pressure < 0.5bar, it is 10kD ultrafiltration and concentration that the film bag finally selected retains aperture, ultra-filtration conditions controls as entrance end pressure < 1.5bar, outlet pressures < 0.5bar, and cycles of concentration controls between 5-10 times.
2, EPO-CTP Blue-FF purifying
Blue Sepharose 6 Fast Flow medium 20mM Tris, 100mM NaCl, pH7.0 ± 0.5; Equilibration buffer 3 times of column volumes (CV), loading is complete uses 20mM Tris, 200mM NaCl, pH7.0 ± 0.5 again; Equilibration buffer 5CV, washes not adsorbable impurity protein off.Finally adopt 20mMTris, 1.2M NaCl, pH7.0 ± 0.5 elution, collect eluted protein peak, the protein solution collected is pressed 10 times of volume computing ultrafiltration desalinations, displacement damping fluid adopts 20mM Tris, pH7.5 ± 0.5.
3, EPO-CTP DEAE-FF chromatography purification
DEAE Sepharose 6 Fast Flow medium 20mM Tris, pH7.5 ± 0.5 equilibration buffer 3CV, loading is complete uses 20mM Tris again, pH7.5 ± 0.5 equilibration buffer 3CV, wash not adsorbable impurity protein off, then use 20mM Tris successively, 50mM NaCl (pH7.5 ± 0.5) and 6M urea+1mM glycine (pH4.1-pH4.3) is wash-out 5CV respectively, removes active low and other impurity protein.Finally adopt 20mM Tris, 150mMNaCl, pH7.5 ± 0.5 elution, collect eluted protein peak.
4, EPO-CTP S-200 chromatography purification
S-200 medium 20mM phosphoric acid buffer+100mM NaCl, pH6.8 ± 0.2 balances 3CV, get the sample loading after DEAE Sepharose 6 Fast Flow purifying, complete 20mM phosphoric acid buffer pH6.8 ± 0.2 of using again of loading balances and wash-out, collects eluted protein peak.By the 0.22um filter membrane Sterile Filtration of the albumen after wash-out.By above method, the Recombinant Human Erythropoietin-CTP fusion rotein that specificity (Fig. 5) purity is greater than 95% (Fig. 4) can be obtained.
Embodiment 5 monkey in vivo bioactivity dynamics research
By subcutaneous injection, EPO-CTP fusion rotein dosage is that 150IU/Kg injects cynomolgus monkey.Before medication and after medication, 0.5h, 2h, 5h, 9h, 24h (D1), 48 (D2), 72 (D3), 96 (D4), 120 (D5), 144 (D6), 168 (D7), 192 (D8), 240 (D10) get whole blood at the forelimb of monkey or hind leg venae subcutaneae and are about 1ml respectively.Adopt ELISA method to measure sample, measure interior to typical curve linear section for diluted sample.After cynomolgus monkey gives EPO-CTP, after medicine, 5h absorbs and reaches peak value, its medicine mean terminal transformation period 12.65h (Fig. 6).
Embodiment 6 is treated rat drug induced injury and is caused pharmacodynamics test in renal anemia body
This experiment, with accurate gavage rat 3 weeks VITAMIN B4 250mg/kg every day, sets up rat drug induced injury animal model of renal anemia.Mould animal is become to be divided into 5 groups at random by HGB level, be respectively dosage group and trial-product high dose group in model control group, the positive (common EPO) control group, trial-product low dose group, trial-product, it is 1350IU/kg that dosage is respectively common EPO, trial-product is low, in and high dosage be respectively 150,450 and 1350IU/kg.And establish Normal group.Experiment 6 groups altogether, often organizes 7, totally 42.Trial-product group Per-Hop behavior 1 time, positive controls administration in every 2 days 1 time, continuous 4 weeks.During administration, every day observes the general clinical symptom of animal, monitors weekly its hematology, blood biochemistry index of correlation, and administration terminates to carry out gross anatomy observation to animal, row histopathologic examination.Test display (Fig. 7) is in the treatment of anaemia, and the middle and high dosage group of Recombinant Human Erythropoietin-CTP fusion rotein and positive controls are improved the effect of anaemia, and it raises RBC, HGB, HCT effect, has dependency with dosage.Recombinant Human Erythropoietin-CTP fusion rotein Per-Hop behavior once, compared with within every two days, being administered once, has long-acting feature with positive controls, reduces administration number of times, improves the conformability for the treatment of.
Embodiment 7 is treated kidney of rats excision damage and is caused pharmacodynamics test in renal anemia body
This experiment employing 5/6 nephrectomy method sets up kidney of rats excision damage animal model of renal anemia.Become mould animal to be divided into 6 groups at random by HGB level, be respectively dosage group and trial-product high dose group in Normal group, model control group, the positive (common EPO) control group, trial-product low dose group, trial-product.Wherein, Normal group 5 animals, all the other each group is 6.It is 450IU/kg that dosage is respectively common EPO, trial-product is low, in and high dosage be respectively 150,450 and 1350IU/kg.Trial-product and negative control are 1 time/week, and positive reference substance is 1 time/2 days, continuously injection 4 weeks.During administration, every day observes the general clinical symptom of animal; Modeling terminates, D8, D15, D22 medicine front and administration terminates monitoring of blood index; Modeling terminates and administration terminates monitoring blood biochemistry index of correlation.Experimental result (Fig. 8) proves that trial-product Recombinant Human Erythropoietin-CTP fusion rotein 450IU/kg, 1350IU/kg and the precious 450IU/kg of positive control drug profit blood is improved the effect of anaemia, in dose-dependently.Trial-product Recombinant Human Erythropoietin-CTP fusion rotein Per-Hop behavior once, compared with within every two days, being administered once, having long-acting feature, which reduces administration number of times, improve the conformability for the treatment of with positive control drug profit blood treasured.
Claims (1)
1. a preparation method for Recombinant Human Erythropoietin-CTP fusion rotein, its step is as follows:
Expression strain construction process: will the EPO-CTP antigen-4 fusion protein gene on pMD18-T carrier be connected to, utilize Not I and EcoR I double digestion qualification positive colony, order-checking, after obtaining the positive colony of correct sequence, be connected to by fusion gene on pcDNA carrier for expression of eukaryon, transfection CHO cell after the expression strain extraction plasmid built, method adopts electricity to transform, the linearizing DNA of 150 μ g adds 5 μ l salmon sperm dnas, CHOK1 host cell 5 × 10
6individual, after mixing, 180V electric shock twice, turns electricity in the plate that rear cell suspension spreads with substratum, treats to occur clone bunch, picking mono-clonal enlarged culturing in plate, through 3 time clonings screenings, obtains relatively stable overexpression cell line,
Zooblast is extensive, high-density, carrier attach perfusion culture: zooblast microcarrier attaches perfusion culture method, mainly comprises the inoculation of cell, initial growth phase, changes culture condition, subsequent growth stage and monitoring culture condition, and concrete steps are as follows:
1) inoculation of cell,
Recover in cell bank one and express EPO-CTP cell strain, add 10% foetal calf serum with DMEM substratum to go down to posterity in T25 square vase three times, adjustment cell state, when cell is in logarithmic phase, with tryptic digestion, increase serum stops digestion, and the DMEM substratum adding 10% foetal calf serum carries out kind of a subchain amplification, cell amplification goes down to posterity step by step according to the order of T25-T75-T150 square vase-2L rolling bottle, inoculate the bio-reactor with microcarrier with the cell of rolling bottle digestion, after inoculation, cell density is 3 × 10
5individual cells/ml,
2) initial growth phase,
After cell access production reactor, set every culture parameters, temperature 37 DEG C, PH6.9-7.2, DO40-70%, rotating speed 40-70 turn/min, and every day, sterile sampling, detected glucose and lactic acid content,
3) culture condition is changed
When glucose content being serum-free medium lower than changing nutrient solution during 1g/L, entering the expression phase, and carrying out filling type cultivation, groundwater increment for keeping glucose content 2g/L,
4) downstream manufacturing stages
When culture cycle reaches 30-40 days, or glucose utilization 24 hours is lower than 0.5g/L, stops cultivating,
5) culture condition is monitored
The index detected is needed to have temperature, pH, glucose concn, lactate concentration, ammonium salt concentration, glutamine concentration, osmolarity, express polypeptide or protein concn in culturing process,
6) purifying
6) 1, purifying pre-treatment
Get cell cultures good harvest liquid 30L, first 4500rpm, 15min, 4 DEG C are centrifugal, then adopt 0.22 μm of form filter element filtering, filter pressure < 0.5bar, it is 10kD ultrafiltration and concentration that the film bag finally selected retains aperture, and ultra-filtration conditions controls as entrance end pressure < 1.5bar, outlet pressures < 0.5bar, cycles of concentration controls between 5-10 times
6) 2, EPO-CTP Blue-FF purifying
Blue Sepharose 6Fast Flow medium 20mM Tris, 100mM NaCl, pH7.0 ± 0.5; Equilibration buffer 3 times of column volumes, loading is complete uses 20mM Tris, 200mM NaCl, pH7.0 ± 0.5 again; Equilibration buffer 5CV, washes not adsorbable impurity protein off, finally adopts 20mM Tris, 1.2M NaCl, pH7.0 ± 0.5 elution, collects eluted protein peak, and the protein solution collected is pressed 10 times of volume computing ultrafiltration desalinations, displacement damping fluid adopts 20mM Tris, pH7.5 ± 0.5
6) 3, EPO-CTP DEAE-FF chromatography purification
DEAE Sepharose 6Fast Flow medium 20mM Tris, pH7.5 ± 0.5 equilibration buffer 3CV, loading is complete uses 20mM Tris again, pH7.5 ± 0.5 equilibration buffer 3CV, wash not adsorbable impurity protein off, then 20mM Tris is used successively, the glycine wash-out 5CV respectively of NaCl and the 6M urea+1mM pH4.1-pH4.3 of 50mM pH7.5 ± 0.5, remove active low and other impurity protein, finally adopt 20mM Tris, 150mMNaCl, pH7.5 ± 0.5 elution, collect eluted protein peak
6) 4, EPO-CTP S-200 chromatography purification
S-200 medium 20mM phosphoric acid buffer+100mM NaCl, pH6.8 ± 0.2 balances 3CV, get the sample loading after DEAE Sepharose 6Fast Flow purifying, complete 20mM phosphoric acid buffer pH6.8 ± 0.2 of using again of loading balances and wash-out, collect eluted protein peak, by the 0.22 μm of filter membrane Sterile Filtration of the albumen after wash-out, by above method, obtain the Recombinant Human Erythropoietin-CTP fusion rotein that specificity purity is greater than 95%.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1422870A (en) * | 2001-12-03 | 2003-06-11 | 第一制糖株式会社 | Fusional protein with intensified activity of internal red-blood-cell formation element |
| WO2004099396A1 (en) * | 2003-05-09 | 2004-11-18 | Crucell Holland B.V. | Cultures of e1-immortalized cells and processes for culturing the same to increase product yields therefrom |
| WO2008056961A1 (en) * | 2006-11-10 | 2008-05-15 | Boryung Pharmaceutical Co., Ltd | A novel fusion protein, cell lines expressing the same and preparation method thereof |
| CN102010473A (en) * | 2010-11-10 | 2011-04-13 | 曹鹏 | Recombinant oxyntomodulin (OXM) fusion protein, and preparation and application thereof |
-
2011
- 2011-09-08 CN CN201110265828.6A patent/CN102994547B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1422870A (en) * | 2001-12-03 | 2003-06-11 | 第一制糖株式会社 | Fusional protein with intensified activity of internal red-blood-cell formation element |
| WO2004099396A1 (en) * | 2003-05-09 | 2004-11-18 | Crucell Holland B.V. | Cultures of e1-immortalized cells and processes for culturing the same to increase product yields therefrom |
| WO2008056961A1 (en) * | 2006-11-10 | 2008-05-15 | Boryung Pharmaceutical Co., Ltd | A novel fusion protein, cell lines expressing the same and preparation method thereof |
| CN102010473A (en) * | 2010-11-10 | 2011-04-13 | 曹鹏 | Recombinant oxyntomodulin (OXM) fusion protein, and preparation and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 促红素对人血小板活动度与血红蛋白水平的影响;马曙轩等;《中国生物制品学杂志》;20030325;第16卷(第03期);165-167,174 * |
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