CN102268402B - Serum free medium and culture method for high expression of erythropoietin in CHO (Chinese hamster ovary) cells - Google Patents
Serum free medium and culture method for high expression of erythropoietin in CHO (Chinese hamster ovary) cells Download PDFInfo
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Abstract
The invention discloses a serum free medium for expressing erythropoietin in CHO (Chinese hamster ovary) cells, and a culture method for high expression of erythropoietin in CHO cells. The serum free medium contains additives D-glucose and sodium butyrate, and also contains one or more of D-galactose, D-mannitose and N-acetylglucosamine. The culture method comprises the steps of: performing serum culture on activated seed cells by using a serum medium, and then performing serum free culture by the serum free medium. By adopting the medium and culture method disclosed by the invention, recombinant human erythropoietin protein of high and stable expression can be obtained, and the glycosylation degree of erythropoietin is improved, i.e. the specific gravity of EPO (erythropoietin) sialic acid is improved. As high as 1.0*107IU recombinant human erythropoietin can be obtained from one liter of culture fluid by the culture method provided by the invention, the expression is stable, and the culture method is simple and suitable for large-scale production.
Description
Technical field
The present invention relates to the animal cell culture field, particularly relate to a kind of serum free medium of expressing cho cell erythropoietin and cultural method that Chinese hamster ovary celI efficiently expresses erythropoietin of being used for.
Background technology
(erythropoietin, Erythropoietin are a kind of glycoprotein analog hormones that stimulates marrow hemopoiesis EPO) to erythropoietin, come to light early than 1906.It is mainly derived from kidney (deriving from liver on a small quantity), and is synthetic by the mesenchymal cell around the cortex pipe.Before recombinant gene was born, EPO mainly extracted from anaemia patient's urine and sheep blood, and yield is very low, and extremely unstable, and physics and chemistry and biological property are difficult to mensuration, also can't large-scale application.The Application of DNA recombinant technology is gene constructed in Chinese hamster ovary cell (Chinese hamster ovary celI) with erythropoietin; Form the Chinese hamster ovary celI of efficiently expressing recombinant human erythropoietin gene; Cultivate through cell amplification; Change and express substratum, collect culture supernatant, after separation and purification can prepare purpose gp-EPO.Under the situation of the Chinese hamster ovary celI strain that obtains expression EPO, the use of substratum has material impact to expression amount and the glycosylation stability of EPO.The EPO expression amount of conventional cell culture medium is about 4000IU/ml, and the EPO expression amount of different substratum is unstable, and the degree of glycosylation of the EPO that is obtained also exists than big-difference, can not well satisfy the EPO need of industrial production.
Summary of the invention
The objective of the invention is to satisfy the problem of high efficiency stable expression EPO in the Chinese hamster ovary celI to present substratum; A kind of serum free medium that can be used for improving EPO expression amount and expression stability is provided, and said serum free medium efficiently expresses the application in the erythropoietin at Chinese hamster ovary celI.
Another object of the present invention provides the said serum free medium of a kind of employing, in Chinese hamster ovary celI, efficiently expresses the cultural method of erythropoietin.
To achieve these goals, the present invention has adopted following technical scheme:
The invention discloses a kind of serum free medium, comprise basic medium and additive, it is characterized in that: said additive comprises butanic acid sodium, also comprises in D-semi-lactosi, D-seminose, the N-acetylglucosamine one or more.
Further, said additive also comprises D-glucose.
Further, the preferred additives consumption is D-semi-lactosi 200-300mg/L among the present invention, D-seminose 200-300mg/L, N-acetylglucosamine 200-300mg/L, D-glucose 1-3g/L, butanic acid sodium 1-2mmol/L.
Said basic medium comprises DMEM substratum and the CHO-S-SFM II substratum of portion rate 1-3: 7-9, and among the present invention, preferred portion rate is 1: 9.
Preferably, said basic medium is made up of DMEM substratum and the CHO-S-SFM II substratum of portion rate 1-3: 7-9, and preferred portion rate is 1: 9.
The invention also discloses a kind of above-mentioned substratum and efficiently express the application in the erythropoietin at Chinese hamster ovary celI.
The invention also discloses the cultural method that efficiently expresses erythropoietin in a kind of Chinese hamster ovary celI; Said cultural method comprises that the employing of the elder generation of the seed cell after the activation is contained blood serum medium contains the serum cultivation, adopts above-mentioned serum free medium to carry out serum-free culture then.
The said blood serum medium that contains is the DMEM substratum that contains mass fraction 5%-10% Ox blood serum, preferred Ox blood serum content 10%.
Saidly contain the MTX that blood serum medium also comprises 0.5-1 μ mol/L.
The said condition that contains serum cultivation and serum-free culture is dissolved oxygen concentration 30%-80%, pH value 7.0-7.4,35 ℃-38 ℃ of temperature.It is pointed out that in the preferred embodiment of the invention that in order to guarantee culture effect, preferred cell inoculation density is greater than 3 * 10
5Cells/mL.
Because adopt above technical scheme, beneficial effect of the present invention is:
Adopt substratum of the present invention and cultural method, make the CHO-EPO cell can be efficiently, stably express recombinant human erythropoietin albumen, improve the degree of glycosylation of cell expressing and target protein, promptly improved the proportion of sialyl EPO.Adopt every liter of nutrient solution of cultural method provided by the invention to obtain up to 1.0 * 10
7The recombinant human erythropoietin of IU, expression is stable, cultural method is succinct, be fit to scale operation.
Description of drawings
Fig. 1 is a cell cultures schema in the embodiment of the invention;
Fig. 2 is the influence result of the butanic acid sodium of different concns in the embodiment of the invention to the EPO expression amount;
Fig. 3 is that different concns MTX influences the result to the EPO expression amount in the embodiment of the invention;
Fig. 4 be in the embodiment of the invention glycosylation reagent to the result that influences of the proportion of high sialic acid EPO;
Fig. 5 is serum-free culture fate and the EPO expression amount of corresponding every day in the embodiment of the invention;
Fig. 6 is serum-free culture fate and corresponding consumption sugar amount every day in the embodiment of the invention.
Embodiment
The invention provides a kind of new serum free medium that can be used for improving EPO expression amount and expression stability, said serum free medium comprises basic medium and additive.Said additive comprises butanic acid sodium, also comprises in D-semi-lactosi, D-seminose, the N-acetylglucosamine one or more, further also comprises D type glucose (D-glucose).Said basic medium comprises the DMEM substratum and the CHO-S-SFM II substratum of certain umber ratio, and among the present invention, preferred basic medium is made up of DMEM substratum and CHO-S-SFM II substratum.Said certain umber ratio is 1-3: 7-9, is preferably 1: 9.Further, the consumption of said additive is D-semi-lactosi 200-300mg/L, D-seminose 200-300mg/L, N-acetylglucosamine 200-300mg/L, D-glucose 1-3g/L, butanic acid sodium 1-2mmol/L.
The present invention also provides above-mentioned substratum to efficiently express application and cultural method in the erythropoietin at Chinese hamster ovary celI, comprising: the employing of the elder generation of the seed cell after the activation is contained blood serum medium cultivate, adopt above-mentioned substratum to carry out serum-free culture then.The concrete flow process of cultivating comprises: seed cell activation amplification cultivation is inoculated into bio-reactor then and contains serum cultivation and serum-free culture respectively, last harvested cell nutrient solution (Fig. 1).Further, also need the pair cell nutrient solution to carry out routine operations such as follow-up separation, purification of target albumen in order to obtain the EPO product.Wherein, (1) seed cell activation amplification cultivation: cell recovery, amplification cultivation is carried out in the DMEM substratum that adds 0.5-1 μ mol/L MTX and 10% Ox blood serum; Use trypsin digestion and cell then, the preparation cell suspension is used to inoculate bio-reactor.(2) containing serum cultivates: in the DMEM substratum that adds 0.5-1 μ mol/L MTX and 10% Ox blood serum, carry out, the control culture condition is dissolved oxygen 30%-80%, pH value 7.0-7.4, and 35 ℃-38 ℃ of temperature are preferably 36.8 ± 0.2 ℃.(3) serum-free culture: the serum free medium with adding 250mg/L D-semi-lactosi, 250mg/L D-seminose, 250mg/L N-acetylglucosamine, 1-3g/L glucose and 1-2mmol/L butanic acid sodium continues culturing cell; The control culture condition is dissolved oxygen 30%-80%; PH value 7.0-7.4; 35 ℃-38 ℃ of temperature are preferably 36.8 ± 0.2 ℃.Said serum free medium is the substratum that described DMEM substratum of the invention described above and CHO-S-SFM II substratum combine.Said seed cell is the Chinese hamster ovary celI that has made up the EPO expression of erythropoietin gene; Can obtain through commercial sources; The general EPO that obtains through commercial sources expresses the screening of Chinese hamster ovary celI process and preserves and handle, and need just can be used for subsequent operations through steps such as overactivations.
Below through specific embodiment and combine accompanying drawing that the present invention is done further explain.Following examples are only further explained the present invention, should not be construed as limitation of the present invention.
According to cultivating flow process; With seed cell activation amplification cultivation, use trypsin digestion and cell then, the preparation cell suspension; Inoculation; After in not containing the 10% Ox blood serum DMEM substratum of MTX, containing the serum cultivation, cell is cultivated respectively at containing in 0mmol/L butanic acid sodium, 0.5mmol/L butanic acid sodium, 1.0mmol/L butanic acid sodium and the 2.0mmol/L serum free medium, said serum free medium comprises 1: 9 DMEM substratum of portion rate and CHO-S-SFM II substratum.36.8 cultivate after 48 hours and collect supernatant for ± 2 ℃, measure the EPO expression amount of culture supernatant respectively.The result shows, the butanic acid sodium of 1-2mmol/L can be stablized the expression amount of unit cell EPO, and the growth of pair cell is not significantly influence also.The result sees Fig. 2.
According to cultivating flow process; With seed cell activation amplification cultivation, use trypsin digestion and cell then, the preparation cell suspension; Inoculation; Cell respectively at after containing the serum cultivation in the 10% Ox blood serum DMEM substratum that adds 0 μ mol/L, 0.5 μ mol/L and 1 μ mol/L MTX, is added in the serum free medium that contains 1.0mmol/L butanic acid sodium respectively and carries out serum-free culture, and said serum free medium comprises that portion rate is 1: 9 DMEM substratum and a CHO-S-SFM II substratum.36.8 cultivate after 48 hours and collect supernatant for ± 2 ℃, measure culture supernatant EPO expression amount respectively.The result shows, adds 0.5-1 μ mol/L MTX in the substratum, can stablize the expression amount of unit cell EPO, and the growth of pair cell is not significantly influence also.The result sees Fig. 3.
According to cultivating flow process; With seed cell activation amplification cultivation; Use trypsin digestion and cell then; The preparation cell suspension, inoculation, and after in the 10% Ox blood serum DMEM substratum of 1 μ mol/L MTX, containing the serum cultivation; Respectively at cultivating in the serum free medium that does not contain glycosylation reagent and the glycosylation reagent that contains D-semi-lactosi 250mg/L, D-seminose 250mg/L, N-acetylglucosamine 250mg/L, said serum free medium comprises 1: 9 DMEM substratum of portion rate and CHO-S-SFM II substratum with cell.36.8 cultivate after 48 hours and collect supernatant for ± 2 ℃, measure the EPO expression amount of culture supernatant respectively.The result shows that glycosylation reagent can increase the proportion of sialyl EPO in the culture supernatant.IEF+Western-blot result sees Fig. 4.
According to cultivating flow process; With seed cell activation amplification cultivation; Use trypsin digestion and cell then, prepare cell suspension, be inoculated into the NBS bio-reactor of 5L; After in the 10% Ox blood serum DMEM substratum of 1 μ mol/L MTX, containing the serum cultivation, in the serum free medium that adds D-semi-lactosi 250mg/L, D-seminose 250mg/L, N-acetylglucosamine 250mg/L, D-glucose 1-3g and 1.0mmol/L butanic acid sodium, will cultivate.Said serum free medium comprises 1: 9 DMEM substratum of portion rate and CHO-S-SFM II substratum.36.8 ± 2 ℃ are cultured to 14 days, the about 60L of cell harvesting liquid, EPO expression amount are 10388IU/mL, results 6.2 * 10
8IU EPO.The high expression level 12878IU/mL of EPO every day, minimum expression 4797IU/mL.EPO expression amount and cell growth condition are seen Fig. 5 and Fig. 6.
Above content is to combine concrete embodiment to the further explain that the present invention did, and can not assert that practical implementation of the present invention is confined to these explanations.For the those of ordinary skill of technical field under the present invention, under the prerequisite that does not break away from the present invention's design, can also make some simple deduction or replace, all should be regarded as belonging to protection scope of the present invention.
Claims (1)
1. efficiently express the cultural method of erythropoietin in the Chinese hamster ovary celI; Concrete steps are following: with seed cell activation amplification cultivation; Use trypsin digestion and cell then; The preparation cell suspension is inoculated into the NBS bio-reactor of 5L, after in the 10% Ox blood serum DMEM substratum of 1 μ mol/L MTX, containing serum and cultivating; In the serum free medium that adds D-semi-lactosi 250mg/L, D-seminose 250mg/L, N-acetylglucosamine 250mg/L, D-glucose 1-3g/L and 1.0mmol/L butanic acid sodium, cultivate; Said serum free medium comprises 1: 9 DMEM substratum of portion rate and CHO-S-SFM II substratum, and 36.8 ± 2 ℃ are cultured to 14 days, the harvested cell nutrient solution; Said seed cell is the Chinese hamster ovary celI that has made up the EPO expression of erythropoietin gene.
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| CN103820514B (en) * | 2014-03-10 | 2017-10-31 | 深圳赛保尔生物药业有限公司 | A kind of method of efficient production hematopoietin |
| CN104099392B (en) * | 2014-07-08 | 2017-01-25 | 苏州康聚生物科技有限公司 | Industrialized production method for high galactosylated modification recombination human erythrocyte growth stimulation protein |
| CN104130971B (en) * | 2014-08-12 | 2015-03-11 | 山西威奇达光明制药有限公司 | Serum-free medium of recombinant human erythropoietin, applications of serum-free medium and method for preparing recombinant human erythropoietin |
| CN105820232B (en) * | 2016-04-08 | 2019-05-17 | 昂德生物药业有限公司 | The preparation method and its product of mono-modified polyethylene glycol Recombinant Human Erythropoietin and application |
| CN109385401A (en) * | 2017-08-10 | 2019-02-26 | 北京泰德制药股份有限公司 | A kind of cultural method of Chinese hamster ovary celI that expressing recombinant protein |
| CN114317446B (en) * | 2021-12-30 | 2023-12-05 | 广州康盛生物科技股份有限公司 | Additive for serum-free culture medium and application thereof |
| CN114561364A (en) * | 2022-03-02 | 2022-05-31 | 上海荣盛生物药业股份有限公司 | Method and reagent for efficiently amplifying human influenza virus |
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