CN1058992C - Recombinative human thrombocytopoietic factor and production thereof - Google Patents
Recombinative human thrombocytopoietic factor and production thereof Download PDFInfo
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
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Abstract
The present invention discloses a production technology for the mass obtainment of human thrombocytopoietic factors (rhTPO) and preparations thereof by a base group engineering method. In the stage of host cell amplification culture, the inoculum concentration of cells is from 1.5*10<4>/cm<2> to 2.5*10<4>/cm<2>, the content of calf serums is from 8 to 10%, cell adherence time is shortened to shorter than 72 hours, a single layer grows, and the stay time of a single layer cell is prolonged to 20 to 24 days, so the secretory expression quantity of a CHO cell to TPO is increased, and consequently, the yield of the TPO is considerably increased. RhTPO is separated from the obtained substance of culture and is purified by the three steps of anion-exchange chromatography, hydrophobic chromatography and molecular sieve gel chromatography. The present invention simplifies the processes of separation and purification, shortens the time of separation and purification, avoids the action of target protein in a polarity condition and increases the stability of the TPO. The yield of a final product is increased from about 30% of a conventional method to about 45%. A proper amount of human serum albumin as a protectant and 1% mannitol are added to the TPO final product produced by the method to prepare an injection which can be used for treating thrombocytopenia caused by various reasons.
Description
The present invention relates to the engineered method of a kind of employing, obtain the production technique of human thrombopoietin (rhTPO) in a large number, and with the prepare medicines method of preparation of its product.
Thrombocyte is a kind of important blood ingredient, results from the megalokaryocyte of marrow.Hinder and take place when hemorrhage at human organ, tissue damaged, thrombocyte is the most important factor that forms local blood clot.As aleukia, the position of human body damaged can not form blood clot, and perhaps blood coagulation is slow, hemorrhage can not stopping and ischemia even threat to life take place.The methods of treatment of aleucia is numerous, and is classical, also is that effective means is blood transfusion or thrombocyte infusion.The shortcoming of this method is to make patient be faced with the pathogenic former danger of infecting as hepatitis B virus, hiv virus etc. of haematogenous, and its harm may surpass aleucia itself.Though other composition of blood such as red corpuscle, leukocytic shortage can effectively solve with the way of " component blood transfusion ", and the thrombocyte infusion is everlasting and immune response is promptly taken place after infusion one secondary and make treatment be forced to interrupt.Therefore, stimulate and promote human body self growth thrombocyte to become the effective means of treatment aleucia.
Since 1960's the thrombocyte Study of Mechanism has been confirmed that in all factors of regulating hematopoiesis, that thrombocyte has been generated most important functions is thrombopoietin (Thrombopoietin, abbreviation TPO).Exogenous TPO can effectively promote the animal and human to generate thrombocyte.Therefore, obtain TPO in a large number, become a task of international the world of medicine in recent decades with the method for suitability for industrialized production.
" Human Megakaryopoietin and separation and purification thereof and gene clone method and application " (Chinese patent publication number CN1107891A), " a kind of new thrombopoietin " patent applications such as (Chinese patent publication number CN1127785A) disclose Human Megakaryopoietin (Megakaryopoietin, be called for short MPO) and the engineered upstream technology such as cDNA sequence encoding of thrombopoietin (TPO), and not open or not fully not openly to the method for scale operation.They and disclosed in the world method are aspect processing sequence or the product quality aspect part that all haves much room for improvement of tiring.
The invention provides a kind of method that can be used in the scale operation recombination human recombination, particularly host cell is carried out large scale culturing, expression product is carried out the method for separation and purification.
Another aspect of the present invention is that the recombinant human TPO with purifying makes medicine preparation as activeconstituents, be used for the treatment of with the aleukia is the disease of feature, as aleukia due to aleucia, radiotherapy, the chemotherapy, the toughener that also can be used for bone marrow transplantation, hematoblastic recovery of patient etc. behind the accelerated bone implantation of marrow.
Successively method of the present invention is described as follows by processing step.
It is host cell that the method that the present invention produces thrombopoietin is selected Chinese hamster ovary (CHO) cell for use.Select pSV-dhfr expression plasmid as a token of plasmid and pCDB cotransfection Chinese hamster ovary cell dihydrofolate reductase gene defective (CHO-dhfr-) strain for use, and then the cell strain of screening high efficiency stable expression people TPO.
One, culturing engineering cell strain
The cultivation flow process of rhTPO-CHO-N cell strain is: cell recovery → pre-cultivation → detection → amplification cultivation → serum-free culture → results supernatant.
In the amplification cultivation stage, the cell inoculation amount is 1.5 * 10
4/ cm
2-2.5 * 10
4/ cm
2, calf serum content is 8-10%.Process goes down to posterity for 4-5 time, and when last generation, cell formed individual layer in rolling bottle, cell density was 1.5-2.0 * 10
6Individual cell/cm
2With Hank ' s liquid washing 6 times, enter serum-free culture.The cell attachment incubation time is 20-24 days during serum-free culture.In the serum-free culture stage, be 3-5 days the pitch time of receiving liquid for twice, and the PH of substratum is 7.3, the rolling bottle rotating speed be 12 change/hour, culture temperature is 37 ℃.
Two, separation and purification rhTPO
The present invention is by " anion-exchange chromatography-hydrophobic chromatography-molecular sieve gel filtration chromatography " three step separation and purification rhTPO.
1. concentrate and desalination
Select the crossing current ultrafiltration and concentration for use, molecular weight cut-off is 10,000-100, and 000, cycles of concentration is 30-50 times.Carry out the ice bath cooling in the ultra-filtration process, generation desugar chain, sex change etc. cause the TPO inactivation in ultra-filtration process to reduce.This operation purified product gross activity rate of recovery is about 80%.
2.DEAE-Sepharose F.F anion-exchange chromatography
DEAE-Sepharose F.F ion column is used 10mM Tris-HCl in advance, three column volumes of pH7.0 damping fluid balance, with the flow velocity sample introduction of rhTPO concentrated solution with 15-40ml/min, sample introduction finishes the back with the 5mM acetic acid of 2.5-5.0 times of column volume, the 1mM glycine, 6M urea, the washing of pH3.5-4.5 damping fluid.After the above-mentioned steps washing, with 20-25mM NaCl/10mM Tris-HCl, the pH7.0 damping fluid is washed till post neutral and removes urea simultaneously, uses 100-150mM NaCl/10mM Tris-HCl at last, pH7.0 buffer solution elution TPO protein peak.This step activity recovery is 35-50%.
3.Butyl-Sepharose F.F hydrophobic chromatography
Butyl-Sepharose F.F post 0.5-1.5M (NH
4)
2SO
4/ 10mM Tris-HCl behind three column volumes of pH7.0 damping fluid balance, will contain 1M (NH
4)
2SO
4Above-mentioned TPO elution peak with sample on the flow velocity of 15-25ml/min, sample introduction finish the back with 0.5-1.0M (NH
4)
2SO
4/ 10mM Tris-HCl, pH7.0 damping fluid wash post to A280nm curve and are back to baseline, use 10-30mM Tris-HCl, and TPO albumen is washed in the lyolysis of pH7.0 buffering.This step activity recovery is 85-95%.
4.Sephacryl S-200 H.R gel permeation chromatography
Sephacryl S-200 H.R post (contains 100mM NaCl with citrate buffer, pH6.8-7.0) after the balance, the TPO protein peak that the hydrophobic chromatography desorb is got off is with sample on the flow velocity of 0.5-1.5ml/min, carry out wash-out with above-mentioned citrate buffer, collect the active peak of single TPO.With the degerming of 0.22um filtering with microporous membrane, place 4 ℃ or-70 ℃ of refrigerators preservations.Detect proof through PAGE, HPLC, finished product purity reaches more than 99%.This step activity recovery is more than 90%.
Three, quality controlling means
Match with suitability for industrialized production of the present invention, set up following quality controlling means:
(1) identity inspection: rhTPO is carried out peptide figure, SDS-PAGE, HPLC, Western blot and amino acid separating tests, and the result should be consistent with standard substance.The N-terminal aminoacid sequence of rhTPO should be in full accord with the TPO native sequences.
(2) protein concentration detects: measure rhTPO concentration by Lowry method, UV method.
(3) purity detecting: reduction or non-reduced SDS-PAGE, HPLC measure, and its purity answers 〉=98%.
(4) pyrogen test: carry out the rabbit method by the pharmacopeia regulation and detect.
(5) pollution of nucleic acid detects test: the DNA residual quantity is answered＜the 10pg/ agent.
(6) protein contamination test: the calf serum residual quantity is answered＜0.01% (w/w); Chinese hamster ovary celI albumen residual quantity should＜0.5% (w/w).
(7) microbial contamination test: sterility test is answered asepsis growth; The mycoplasma inspection should not have mycoplasma contamination.
(8) titration: comprise ELISA method (external titration), many blood mouse method (titration in the body).
(9) stability test: by the stability test under the different storage conditions, the validity period of determining finished product is a year and a half.
Produce TPO by method of the present invention, at the cell amplification cultivation stage, the cell attachment time was foreshortened to below 72 hours grow up to individual layer, the monolayer cell residence time extends to 20-24 days, Chinese hamster ovary celI is improved the secreting, expressing amount of TPO, thereby improved the yield of TPO greatly.Use three-step approach of the present invention that culturing cell gained supernatant liquor is carried out separation and purification, then simplified separation purifying technique, shortened the separation and purification time, avoided the effect of target protein under polarity condition, increased the stability of TPO.The yield of finished product is brought up to about 45% from about 30% of ordinary method, promptly improves finished product output about 50%.
The TPO finished product that uses method of the present invention to produce adds an amount of human serum albumin and makes protective material, adds 1% N.F,USP MANNITOL again and is mixed with injection liquid, can be used for the treatment of the thrombocytopenia that a variety of causes causes.
By the following examples content of the present invention is further described.
Clone and the expression of embodiment 1 TPO in Chinese hamster ovary cell (Chinese hamster ovary celI)
Expression vector in Chinese hamster ovary celI has following characteristics:
(1) penbritin (Amp) resistant gene is arranged;
(2) E.coli. replication initiation;
(3) CMV-RSV promotor, heel BamH I and Pvu II cloning site, SV40 intron and polyadenylation site;
(4) little mouse dihydrofolate reductase (mDHFR) gene, this gene performance methotrexate (MTX) resistance.The DHFR genetic transcription is under the control of SV40 promotor.
The dna sequence dna of coding TPO is obtained through the PCR reaction by T1 clone's DNA, 5 '-hold primer to be:
5 '-CCCAGATCTGCCAGAATGGAGCTGAC-3 ' contains Bgl II site.3 '-hold primer to be:
5 '-AAAGATATCTTACCCTTCCTGAGACAG-3 ' contains the complementary sequence of EcoRV site and terminator codon.The PCR product is through Bgl II and EcoRV digestion, and carrier connects then through BamH I and Pvu II digestion.It is XLI Blue that the connection mixture is used for transformed into escherichia coli (E.coli.).Conversion fluid is applied on penbritin (Amp) dish.
Import Chinese hamster ovary celI with the liposome transfection method, and in the no Nucleotide substratum of the MTX that contains 0.02 μ M-0.1 μ Md, select to cultivate.Choosing the MTX resistant cell selects in the ever-increasing substratum of MTX concentration and increases.Choose the highest cell of output, limiting dilution carries out subclone, obtains the ideal clone cell at last.
The amplification cultivation of embodiment 2 rhTPO-CHO cells
The cultivation flow process of rhTPO-CHO-N cell strain is: cell recovery → pre-cultivation → detection → amplification cultivation → serum-free culture → results supernatant.
In the amplification cultivation stage, the cell inoculation amount is 2 * 10
4/ cm
2, calf serum content is 8%.Through serum amplification cultivation (4-5 time go down to posterity) was arranged in 15 days, last in generation cell in rolling bottle, form individual layer (cell density be 1.5-2.0 * 10
6Individual cell/cm
2) time, with Hank ' s liquid washing 6 times, use serum free medium (DMFM/F instead
121: 1) cultivate.The cell attachment incubation time is 20-24 days during serum-free culture, collects supernatant liquor once every 5 days, and liquid volume added is 120ml/ bottle (780cm
2), medium pH is 7.3, the rolling bottle rotating speed be 12 change/hour, culture temperature is 37 ℃.Results are 4 times continuously.RhTPO tires about 1200u/ml.
Embodiment 3 separation and purification rhTPO
Press following three step separation and purification rhTPO:
1. concentrate and desalination: the crossing current ultrafiltration and concentration of selecting for use Millipore company to produce, molecular weight cut-off is 10,000, cycles of concentration is 30 times.Carry out the ice bath cooling in the ultra-filtration process.
2.DEAE-Sepharose F.F anion-exchange chromatography: DEAE-Sepharose F.F ion column is used 10mM Tris-HCl in advance, three column volumes of pH7.0 damping fluid balance, with the flow velocity sample introduction of rhTPO concentrated solution with 35ml/min, sample introduction finishes the back with the 5mM acetic acid of 3 times of column volumes, the 1mM glycine, 6M urea, the washing of pH4.5 damping fluid.The washing back is washed till post neutral and removes urea simultaneously with 25mM NaCl/10mM Tris-HCl, pH7.0 damping fluid, uses 100mM NaCl/10mM Tris-HCl at last, pH7.0 buffer solution elution TPO protein peak.
3.Butyl-Sepharose F.F hydrophobic chromatography: Butyl-Sepharose F.F post 1M (NH
4)
2SO
4/ 10mM Tris-HCl behind three column volumes of pH7.0 damping fluid balance, will contain 1M (NH
4)
2SO
4The TPO elution peak that obtained of above-mentioned steps 2 with sample on the flow velocity of 15ml/min, sample introduction finishes the back with 1M (NH
4)
2SO
4/ 10mMTris-HCl, pH7.0 damping fluid wash post to A280nm curve and are back to baseline, use 10mM Tris-HCl, and TPO albumen is washed in the lyolysis of pH7.0 buffering.
4.Sephacryl S-200 H.R gel permeation chromatography: Sephacryl S-200 H.R post (contains 100mM NaCl with citrate buffer, pH6.8-7.0) after the balance, the TPO protein peak that the hydrophobic chromatography desorb is got off, with sample on the flow velocity of 1.5ml/min, carry out wash-out with above-mentioned citrate buffer, collect the active peak of single TPO.With the degerming of 0.22um filtering with microporous membrane, be work in-process, place 4 ℃ or-70 ℃ of refrigerators preservations.
Detect through PAGE, HPLC, finished product purity can reach more than 99%.
Embodiment 4 preparation for preparing medicines
The TPO finished product that use is produced by the method for embodiment 2 is made activeconstituents, adds an amount of human serum albumin and makes protective material, adds 1% N.F,USP MANNITOL again, is mixed with injection liquid according to a conventional method, can be used for the treatment of the thrombocytopenia that a variety of causes causes.
Claims (1)
1. the method that using gene engineering technique is produced recombination human recombination comprises the steps:
Recovery contains the Chinese hamster ovary cell of recombination human recombination gene;
Cell to recovery is cultivated in advance:
To cultivate the cell culture of gained in advance with 1.5 * 10
4/ cm
2-2.5 * 10
4/ cm
2The cell inoculation amount be inoculated in the substratum that calf serum content is 8-10% and carry out amplification cultivation;
The amplification cultivation thing is cultivated with serum free medium, and wherein the cell attachment time is 20-24 days;
The results culture supernatants;
Supernatant liquor is carried out anion-exchange chromatography, and three steps of hydrophobic chromatography and molecular sieve gel chromatography are separated and the purification of Recombinant human thrombopoietin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN96114273A CN1058992C (en) | 1996-12-25 | 1996-12-25 | Recombinative human thrombocytopoietic factor and production thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN96114273A CN1058992C (en) | 1996-12-25 | 1996-12-25 | Recombinative human thrombocytopoietic factor and production thereof |
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| CN1186119A CN1186119A (en) | 1998-07-01 |
| CN1058992C true CN1058992C (en) | 2000-11-29 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555760A (en) * | 2013-10-18 | 2014-02-05 | 江苏康禾生物制药有限公司 | Preparation method and preparation of recombinant human thrombopoietin |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100415769C (en) * | 2002-02-07 | 2008-09-03 | 中国科学院过程工程研究所 | Method for separation and purification of oligomeric or multimeric subunit proteins |
| CN102309743A (en) * | 2011-08-03 | 2012-01-11 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | New purpose of recombinant human thrombopoietin (rhTPO) |
| CN105541994B (en) * | 2015-12-01 | 2019-12-06 | 沈阳三生制药有限责任公司 | method for purifying thrombopoietin or variant or derivative thereof |
| CN110627892B (en) * | 2019-09-05 | 2023-04-14 | 江苏康禾生物制药有限公司 | Preparation method of recombinant human thrombopoietic factor stock solution |
| CN111893155A (en) * | 2020-07-10 | 2020-11-06 | 深圳市华启生物科技有限公司 | Method for producing recombinant protein |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1127785A (en) * | 1995-04-14 | 1996-07-31 | 李维克 | Human thrombopoietin |
| CN1129805A (en) * | 1995-09-08 | 1996-08-28 | 济南金鲁生物工程有限公司 | Determination of human thrombopoietin variant and its cDNA clone |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1127785A (en) * | 1995-04-14 | 1996-07-31 | 李维克 | Human thrombopoietin |
| CN1129805A (en) * | 1995-09-08 | 1996-08-28 | 济南金鲁生物工程有限公司 | Determination of human thrombopoietin variant and its cDNA clone |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103555760A (en) * | 2013-10-18 | 2014-02-05 | 江苏康禾生物制药有限公司 | Preparation method and preparation of recombinant human thrombopoietin |
| CN103555760B (en) * | 2013-10-18 | 2015-05-20 | 江苏康禾生物制药有限公司 | Preparation method and preparation of recombinant human thrombopoietin |
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| Publication number | Publication date |
|---|---|
| CN1186119A (en) | 1998-07-01 |
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