CN102268407B - Large-scale serum-free culture method for rhIL-12 engineering cells - Google Patents
Large-scale serum-free culture method for rhIL-12 engineering cells Download PDFInfo
- Publication number
- CN102268407B CN102268407B CN201110208009.8A CN201110208009A CN102268407B CN 102268407 B CN102268407 B CN 102268407B CN 201110208009 A CN201110208009 A CN 201110208009A CN 102268407 B CN102268407 B CN 102268407B
- Authority
- CN
- China
- Prior art keywords
- rhil
- serum
- cell
- free
- engineering cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a large-scale serum-free culture method for rhIL-12 engineering cells, which comprises the following step of: inoculating rhIL-12 engineering cells in a logarithmic phase into a serum-free and protein-free medium for culture, wherein the medium is a CD CHO liquid medium comprising sodium pyruvate at the final concentration of 0.8 to 1.2mM, hypoxanthine at the final concentration of 0.075 to 0.125mM, thymidine at the final concentration of 0.012 to 0.020mM, adenosine at the final concentration of 0.5 to 0.9mg/L, guanosine at the final concentration of 0.5 to 0.9mg/L, cytidine at the final concentration of 0.5 to 0.9mg/L, uridine at the final concentration of 0.5 to 0.9mg/L, L-glutamine at the final concentration of 0.4 to 0.8mg/L, L-asparagine at the final concentration of 0.4 to 0.8mg/L, L-proline at the final concentration of 1.5 to 2.0mg/L and non-essential amino acid at the final concentration of 0.08 to 0.125mM. The invention also provides a medium used in the method. By the medium and the culture method, a high-yield and high-activity recombinant human interleukin-12 can be obtained.
Description
Technical field
The invention belongs to biological technical field, relate to cell culture medium and cultural method.Particularly, the present invention is that a kind of Serum-free and protein-free medium that utilizes carries out the method for large scale culturing to rhIL-12(p70) engineering cell, utilize the method to cultivate rhIL-12(p70) engineering cell, can obtain high yield, highly active rhIL-12(p70).
Background technology
(Human Interleukin 12, hIL-12) is a kind of heterodimer cytokine to hIL-12, and Youp35 Hep40Liang Ge subunit connects to form by multipair disulfide linkage.HIL-12 is called again NKSF (NKSF), there is strong antitumor and antiviral benefit, this effect is by the natural killer cell of body (Natural killer cells, NK cell) mediate, hIL-12 also plays an important role in the Development And Differentiation of cytotoxic T cell, helper T cell, B cell and ripe activation, be described as immunity system and exercise antiviral, antineoplastic core regulatory factor, the key protein matter of body natural immune system.This series of biological function makes people recognize that hIL-12 likely plays a significant role in clinical application.At present; rhIL-12(p70) (Recombinant Human Interleukin 12; rhIL-12) as a kind of medicine, entered the clinical experimental stage of antitumor, antiviral property disease and allergic asthma, but still there is certain difficulty in its large-scale production.Therefore, by vitro culture obtain high yield, highly active rhIL-12 has become the problem of needing solution badly.Due to the complicacy of IL-12 structure, make it cannot in E.coli, obtain expression, must adopt eukaryotic expression system to obtain recombinant protein.The heterodimer molecule that IL-12 (p70) molecule is comprised of p40 and two different subunits of p35, two subunits of p40 and p35, respectively by two different genes encodings on two karyomit(e)s, only produce and just can obtain the dimer of biologic activity simultaneously in same cell.The natural expression of p40 and two subunits of p35 is seriously uneven, and in endochylema, the generation of p40 is greater than p35, and the p40 of secretion is easy to form dimer, and the activity of IL-12 is had to restraining effect.Because the imbalance of two chains in born of the same parents expressed, thereby be difficult to obtain the engineering cell strain of high expression level rhIL-12.In addition, rhIL-12 inactivation very easily in preparation process, causes the rate of recovery extremely low, thereby has limited output.At present, the cell for eukaryotic expression mainly contains yeast cell, COS cell, insect cell, Chinese hamster ovary celI.Former three, because of more difficult direct secretion recombinant protein or can not continuous expression protein product or to cultivate cost higher, is difficult to obtain large-tonnage product in practical application.Expressing cho cell system, avoided the deficiency of above-mentioned system, foreign protein is easy to be secreted in substratum, can correctly assemble multimeric protein, exogenous origin gene integrator is stable, can complete posttranslational modification, can reach with suspension culture or in serum free medium high-density, cultivation scale is easy to amplify, and has become one of best host of expression alien gene.Therefore, Chinese hamster ovary celI becomes the first-selected carrier that rhIL-12 produces.Comprehensive above-mentioned reason, through effort in a few years, we have successfully set up the recombinaant CHO cell (rhIL-12 engineering cell) of high efficient expression rhIL-12 albumen, obtained national inventing patent (China Patent No.: zl.03131567.4), for the foundation of rhIL-12 engineering cell large-scale cultivation method is laid a good foundation.
Serum free medium is not containing the substratum of human or animal's serum, but may comprise indivedual albumen or a large amount of protein ingredient, is mainly used in the cell of some specific types that need to grow and for the cultivation of the engineering cell of protein production under serum-free condition.Serum free medium is the important tool that engineering cell is cultivated, and cell cultures is carried out under the condition of a set of restriction, has avoided in serum complicated ingredient on the interference of follow-up study and clinical use and impact.Use serum free medium that engineering cell is cultivated and had the following advantages: (1). increase the determinacy of product; (2). product property is more consistent; (3). easily carry out purifying and Downstream processing; (4). the accurate assessment of cell function; (5). strengthen growth and/or output; (6). the better contrast of physiological response; (7). strengthen the detection of intermediary in cell.The cultivation of reconstitution cell is one of key link of genetically engineered drug research and development.Researchist is devoted to set up the method for easy handling, output is high, its lytic activity is high, cost is low engineering cell large scale culturing always, and wherein bio-reactor accounts for critical role in large scale culturing.Bio-reactor can be divided three classes by cell cultures mode difference: bio-reactor for suspension culture, bio-reactor, embedding culture bio-reactor for adherent culture.Suspension culture does not need to use microcarrier with bio-reactor, and cell is not adherent in reactor, is suspended in cell culture fluid and grows, as stirred reactor, hollow fiber reactor, airlift reactor etc.Adherent culture need to be used microcarrier with bio-reactor, and microcarrier suspension is in the cell culture fluid of reactor, and cell is attached on microcarrier grows, as stirred reactor (microcarrier cultivation), hollow fiber reactor etc.Embedding culture is used porous support or micro-capsule with bio-reactor, and cell is trapped within carrier or is embedded in micro-capsule, as fluidized-bed reactor, fixed bedreactor.U.S. NBS (New Brunswick Scientific Co., inc) bio-reactor that company produces is exactly a kind of embedding culture bio-reactor, its advantage is at utmost to reduce the injury of stirring shearing force to cell, the easy incubation growth of cell, both can cultivate for attached cell, can be used for again suspension cell culture.
In order to reduce clinical application risk and to be convenient to downstream purification process, consider, we are cultivating rhIL-12 engineering cell containing in the substratum of 10% serum, reduce gradually the serum content in substratum, finally arrive serum-free culture, carried out multiple exploration, finally selected CD CHO serum free medium as production substratum, rhIL-12 engineering cell can be grown and hypersecretion IL-12 in the CD CHO serum free medium that limits chemical composition, qualified (report number: SH201000431) is rechecked by three grades of seed bank Bing Jing Nat'l Pharmaceutical & Biological Products Control Institute that set up engineering cell.We have explored rhIL-12 engineering cell and have been amplified to the method that fermentor tank scale is cultivated, the technical process of having set up the extensive serum-free culture of rhIL-12 engineering cell strain from static cultivation simultaneously.Before the present invention, some researchists have carried out that respectively the gene of p40 and p35 is inserted respectively to same carrier and have carried out cotransfection and (wear swallow, Chen Weifeng. the expression of people's recombinant interleukin 12 in Chinese hamster ovary celI. Progress in Biochemistry and Biophysics, 1998, 25 (3): 254-258), or the gene fusion structure IL-12 strand carrier for expression of eukaryon of p40 and p35 is expressed to (Zhang Mei, department carries out raw, Jin Li etc. the structure of recombined human single-chain interleukin 12 fusion genes, eukaryotic expression and Determination of biological activity. Chinese Journal of Hematology, 2000, 21 (12): 636-640), or by transfection hIL-12 carrier cell carry out cellar culture (Liu Meng, Zheng Shanshan. recombinant human cytokine 12 genes are at CHO-dhfr
-the stably express of cell. Journal of Immunology, 1996,12 (4): 215-219), but all fail to realize carrier cell high efficient expression rhIL-12 in large-scale serum-free culture situation.The technique that application the present invention sets up is carried out large scale culturing to rhIL-12 engineering cell, and serum-free culture supernatant is identified through Western blot, at 40kD and 35kD place, occurred band of expression.Serum-free culture supernatant has the ability that promotes NKG cell to produce IFN-γ, and its activity of comparing with international standard substance is in (4-6) * 10
6between IU/mg, more than the content of rhIL-12 (p70) can reach 5mg/L, perfusion is 60 days continuously.With the calculating of working of 2 7.5L fermentor tanks simultaneously, monthly can produce approximately containing the serum-free culture supernatant of 3000mg rhIL-12 (p70) albumen, can meet the requirement of suitability for industrialized production.
Summary of the invention
Therefore, the invention provides pressurization substratum, production Serum-free and protein-free medium and the stable rhIL-12 engineering cell strain large scale culturing technique of screening rhIL-12 engineering cell.
Particularly, one aspect of the present invention provides a kind of rhIL-12 engineering cell extensive serum-free culture method, comprises the steps:
RhIL-12 engineering cell in logarithmic phase is inoculated in Serum-free and protein-free medium and is cultivated, described substratum is to contain the Sodium.alpha.-ketopropionate that final concentration is respectively 0.8-1.2mM, the xanthoglobulin of 0.075-0.125mM, the Thymine deoxyriboside of 0.012-0.020mM, the adenosine of 0.5-0.9mg/L, the guanosine-of 0.5-0.9mg/L, the cytidine(C of 0.5-0.9mg/L, the uridine of 0.5-0.9mg/L, the L-glutaminate of 0.4-0.8mg/L, the altheine of 0.4-0.8mg/L, the L-PROLINE of 1.5-2.0mg/L, the CD CHO liquid nutrient medium of the non-essential amino acid of 0.08-0.125mM.Can be used for rhIL-12 engineering cell of the present invention can build and cultivate according to any methods known in the art, also the application number that can submit to by May 27th, 2003 is 03131567.4, and the description of the Chinese patent application specification sheets that publication number is CN1467292A obtains.
In one embodiment of the invention, the extensive serum-free culture method of rhIL-12 engineering cell of the present invention is carried out in fermentor tank.
In one embodiment of the invention, the remaining sugar concentration preferably maintaining in described substratum is about 10mmol/L.
In another embodiment of the invention, preferably maintain remaining sugar concentration in described substratum more than 10mmol/L, and control lactic acid concn below 20mmol/L.
In one embodiment of the invention, described rhIL-12 engineering cell can obtain by pressurization Screening of Media.Preferably, described pressurization substratum be contain that final concentration is respectively the Sodium.alpha.-ketopropionate of 1.0mM, the Thymine deoxyriboside of the xanthoglobulin of 0.1mM, 0.016mM, the guanosine-of the adenosine of 0.7mg/L, 0.7mg/L, the uridine of the cytidine(C of 0.7mg/L, 0.7mg/L, the altheine of the L-glutaminate of 0.6mg/L, 0.6mg/L, the CD CHO liquid nutrient medium of the non-essential amino acid of the L-PROLINE of 1.7mg/L, 0.1mM, 500 methionine sulfoxides of μ M, the G418 of 800mg/L.
In the extensive serum-free culture method of rhIL-12 engineering cell of the present invention, preferably also comprise the step that perfusion is cultivated.
In one embodiment of the invention, method of the present invention comprises the step of collecting and preserving the rhIL-12 in described substratum supernatant.Described preservation can be-20 ℃ of preservations.Preferably, method of the present invention also comprises the step being further purified.
The rhIL-12 content that the method according to this invention obtains can be more than 5mg/L, and wherein in rhIL-12, the ratio of p40 and p35 is about 1: 1.
In one embodiment of the invention, the invention provides a kind of extensive serum-free culture method of rhIL-12 engineering cell, comprise the steps:
1) the static cultivation of engineering cell: recovery rhIL-12 engineering cell is from working cardial cell storehouse, is inoculated in pressurization substratum and cultivates;
2) amplification cultivation of engineering cell: the rhIL-12 engineering cell in logarithmic phase is inoculated in culture bag and is cultivated;
3) serum-free culture of engineering cell in fermentor tank:
3.1) in fermentor tank, add production serum free medium, inoculating cell;
3.2) perfusion is cultivated: the variation of monitoring at any time dissolved oxygen, glucose, lactic acid and ammonium ion concentration after inoculating cell, according to the variation of oxygen consumption rate, glucose consumption, lactic acid concn, ammonium ion concentration and rhIL-12 albumen generation, carry out perfusion, flow is 0.5-1.5 tank volume;
3.3) collection of serum-free culture supernatant and preservation: when adding production with Serum-free and protein-free medium in perfusion mode, collect culture supernatant in appropriate containers ,-20 ℃ of preservations;
3.4) concentration of the rhIL-12 in serum-free culture supernatant and active detection: use ELISA method to detect the concentration of rhIL-12 in culture supernatant, the balance that in use Western blot method evaluation culture supernatant, p40 and p35 express, and detect the activity of rhIL-12 albumen in culture supernatant, promote NKG cell to produce the ability of IFN-γ.
The present invention also provides a kind of rhIL-12 engineering cell Serum-free and protein-free medium on the other hand, described substratum is to contain the Sodium.alpha.-ketopropionate that final concentration is respectively 0.8-1.2mM, the xanthoglobulin of 0.075-0.125mM, the Thymine deoxyriboside of 0.012-0.020mM, the adenosine of 0.5-0.9mg/L, the guanosine-of 0.5-0.9mg/L, the cytidine(C of 0.5-0.9mg/L, the uridine of 0.5-0.9mg/L, the L-glutaminate of 0.4-0.8mg/L, the altheine of 0.4-0.8mg/L, the L-PROLINE of 1.5-2.0mg/L, the CD CHO liquid nutrient medium of the non-essential amino acid of 0.08-0.125mM.
Preferably, substratum provided by the invention and cultural method are as described below:
1. screen the pressurization substratum of rhIL-12 engineering cell: CD CHO liquid nutrient medium, containing Sodium.alpha.-ketopropionate (0.1mM), xanthoglobulin (0.1mM), Thymine deoxyriboside (0.016mM), adenosine (0.7mg/L), guanosine-(0.7mg/L), cytidine(C (0.7mg/L), uridine (0.7mg/L), L-glutaminate (0.6mg/L), altheine (0.6mg/L), L-PROLINE (1.7mg/L), non-essential amino acid (0.1mM), methionine sulfoxide (500 μ M), G418 (800mg/L).In bracket, it is the final concentration of this material.
2. the Serum-free and protein-free medium that is applicable to the growth of rhIL-12 engineering cell hypersecretion IL-12: CD CHO liquid nutrient medium, containing Sodium.alpha.-ketopropionate (0.8-1.2mM), xanthoglobulin (0.075-0.125mM), Thymine deoxyriboside (0.012-0.020mM), adenosine (0.5-0.9mg/L), guanosine-(0.5-0.9mg/L), cytidine(C (0.5-0.9mg/L), uridine (0.5-0.9mg/L), L-glutaminate (0.4-0.8mg/L), altheine (0.4-0.8mg/L), L-PROLINE (1.5-2.0mg/L), non-essential amino acid (0.08-0.125mM).In bracket, it is the final concentration of this material.
The technical process of 3.rhIL-12 engineering cell large scale culturing
The static cultivation of 3.1 engineering cells: (liquid nitrogen container) recovery rhIL-12 engineering cell is one from working cardial cell storehouse, and with (1-2) * 10
5the density of/ml is inoculated in pressurization substratum, uses 250ml culturing bottle, and 37 ℃, 5%CO
2, cultivate.
The amplification cultivation of 3.2 engineering cells: by the rhIL-12 engineering cell in logarithmic phase after aseptic Hanks washs (800rpm, 10min, 25 ℃) twice, with (2-5) * 10
4the density of/ml is inoculated in and 2000ml production is housed with in 10 liters of big or small culture bag of Serum-free and protein-free medium.37 ℃, 5.0%CO
2, CO
2flow velocity: 0.12-0.18L/min, 6-10rpm/6 °, cultivates.
3.3 the serum-free culture of engineering cell in fermentor tank:
3.3.1 fermentor tank parameter setting: after using sterile purified water that fermentor tank is rinsed well, add the aseptic PBS solution of 5L, proofread and correct pH electrode, connect various gas ducts, stirring velocity 150rpm, is full of water by jacket layer, after machine operates steadily, 121 ℃, 20min autoclaving.Again proofread and correct pH electrode, PBS solution is pumped, add 5L production serum free medium, 50rpm, 37 ℃, logical pressurized air, move after 24 hours.Proofread and correct dissolved oxygen electrode, take final concentration as (3-5) * 10
5/ ml inoculating cell, adjusts parameter: rotating speed is that 50rpm-120rpm, temperature are 36 ℃-37 ℃, and dissolved oxygen is that 30%-60%, pH value be 6.90-7.20, and temperature adopts PID automatic control mode, gas control employing dissolved oxygen-pH master mode that links.
3.3.2 perfusion is cultivated: the variation of monitoring at any time dissolved oxygen, glucose, lactic acid and ammonium ion concentration after inoculating cell, according to the variation of oxygen consumption rate, glucose consumption, lactic acid concn, ammonium ion concentration and rhIL-12 (p70) albumen generation etc., carry out perfusion, flow is 0.5-1.5 tank volume.
The collection of 3.4 serum-free culture supernatants and preservation: when adding production with Serum-free and protein-free medium in perfusion mode, collect culture supernatant in appropriate containers, 12000g, 4 ℃, centrifugal 30 minutes, centrifugal rear supernatant is packed in 1000ml medium bottle,-20 ℃ of preservations, to be purified.
The concentration of rhIL-12 (p70) in 3.5 serum-free culture supernatants and active detection: use ELISA method to detect the concentration of rhIL-12 (p70) in culture supernatant, the balance that in use Western blot method evaluation culture supernatant, p40 and p35 express, and detect the activity of rhIL-12 albumen in culture supernatant, promote NKG cell to produce the ability of IFN-γ.
Accompanying drawing explanation
The grown form (200 *) of Fig. 1 .rhIL-12 engineering cell in Serum-free and protein-free medium.
The growth velocity of Fig. 2 .rhIL-12 engineering cell in Tissue Culture Flask.
Fig. 3 .rhIL-12 engineering cell produces the ability of rhIL-12 (p70) albumen in Tissue Culture Flask.
Fig. 4 .rhIL-12 engineering cell produces the ability of IL-12 (p70) albumen in different serum free mediums.
Note: rhIL-12 engineering cell in CD CHO substratum rhIL-12 (p70) protein secretion apparently higher than other serum free mediums, p < 0.05.
The rate of propagation of Fig. 5 .rhIL-12 engineering cell in culture bag.
Fig. 6 .rhIL-12 engineering cell produces the ability of rhIL-12 (p70) albumen in culture bag.
Fig. 7. the relation of rhIL-12 (p70) protein concentration in remaining sugar concentration and culture supernatant in fermentor tank.
Fig. 8. the relation of remaining sugar concentration and lactic acid concn in fermentor tank.
Fig. 9. the variation of rhIL-12 in fermentor cultivation process (p70) protein concentration, lactic acid concn, ammonium ion concentration and oxygen uptake rate.
Figure 10. the expression of rhIL-12 albumen p35, p40 subunit in fermentor cultivation supernatant.
Embodiment
Embodiment: the screening of the rhIL-12 engineering cell of serum-free suspended state growth, is suitable for the pressurization substratum of rhIL-12 engineering cell growth, produce the foundation by the selection of serum free medium and stable rhIL-12 engineering cell large scale culturing technique
One, material
RhIL-12 engineering cell (obtaining according to the description of China Patent Publication No. CN1467292A), former substratum is the DMEM substratum containing 10% new-born calf serum.DMEM liquid nutrient medium, CHO III A liquid nutrient medium, CD CHO A liquid nutrient medium, CD CHO liquid nutrient medium and SFM liquid nutrient medium are all purchased from Invitrogen company; HyQ SFX-CHO serum free medium is purchased from Hyclone company.100 * non-essential amino acid, 100 * Sodium.alpha.-ketopropionate and 100 * HT are purchased from Invitrogen company; Adenosine, guanosine-, cytidine(C, uridine and thymidine are purchased from BioBASic company; L-glutaminate, altheine and L-PROLINE are purchased from Sigma company; New-born calf serum (super) is purchased from Shanghai Excell Biology Product Co., Ltd.; The sterile filters in 0.22 μ m aperture is purchased from Millipore company; Dimethyl sulfoxide (DMSO), trypsinase are purchased from Shanghai Sheng Gong biotechnology company limited; G418 is purchased from Invitrogen company; Methionine sulfoxide is purchased from Sigma company; Human IL-12 (p70) ELISA detection kit and Human IFN-γ ELISA detection kit are purchased from Bender company.
Two, key instrument equipment
Micropipet is purchased from French Gilson company, and 78-1 type magnetic force heating stirrer is purchased from Jiangsu An Pu electronic engineering company limited.Clean work station is purchased from Jinan City, Shandong Province air purifying and sterilizing instrument factory, and AvantiTM J-20 * P type whizzer is purchased from Beckman company, and Ultralow Temperature Freezer (70 ℃) is purchased from Harris Manufacturing CO. company, CO
2incubator is purchased from ThermoForme company, and microplate reader is purchased from BIO-RAD company.Wave bio-reactor (2/10 type) and culture bag are purchased from U.S. Wave Biotech company; 7.5L fermentor tank is purchased from U.S. NBS company.
Three, experimental technique and result
1. the preparation of DMEM liquid nutrient medium completely: get DMEM liquid nutrient medium 1000ml, add respectively following material, it in bracket, is the final concentration of this material: Sodium.alpha.-ketopropionate (1.0mM), xanthoglobulin (0.1mM), Thymine deoxyriboside (0.016mM), adenosine (0.7mg/L), guanosine-(0.7mg/L), cytidine(C (0.7mg/L), uridine (0.7mg/L), L-glutaminate (0.6mg/L), altheine (0.6mg/L), L-PROLINE (1.7mg/L), non-essential amino acid (0.1mM), new-born calf serum (10%, v/v).
2. the preparation of pressurization substratum: get CD CHO liquid nutrient medium 1000ml, add respectively following material, it in bracket, is the final concentration of this material: Sodium.alpha.-ketopropionate (1.0mM), xanthoglobulin (0.1mM), Thymine deoxyriboside (0.016mM), adenosine (0.7mg/L), guanosine-(0.7mg/L), cytidine(C (0.7mg/L), uridine (0.7mg/L), L-glutaminate (0.6mg/L), altheine (0.6mg/L), L-PROLINE (1.7mg/L), non-essential amino acid (0.1mM), methionine sulfoxide (500 μ M), G418 (800mg/L).
3. produce by Serum-free and protein-free medium (I, II, III) preparation: get CD CHO liquid nutrient medium 1000ml, add respectively following material, it in bracket, is the final concentration of this material: Sodium.alpha.-ketopropionate (0.8-1.2mM), xanthoglobulin (0.075-0.125mM), Thymine deoxyriboside (0.012-0.020mM), adenosine (0.5-0.9mg/L), guanosine-(0.5-0.9mg/L), cytidine(C (0.5-0.9mg/L), uridine (0.5-0.9mg/L), L-glutaminate (0.4-0.8mg/L), altheine (0.4-0.8mg/L), L-PROLINE (1.5-2.0mg/L), non-essential amino acid (0.08-0.125mM).
4. the screening of the rhIL-12 engineering cell of serum-free suspended state growth:
4.1 experimental programs: progressively reduce the serum content for rhIL-12 engineering cell DMEM substratum, by 10% to 5% again to 1%, every kind of serum-concentration is cultivated 12-14 days, visual cell's growing state goes down to posterity during this time, use afterwards CD CHO pressurization substratum to replace DMEM substratum by different ratios (by 50% to 75% again to 100%), every kind of ratio maintain 12-14 days, visual cell's growing state goes down to posterity during this time, after rhIL-12 engineering cell is suspended state growth, uses limiting dilution assay to carry out mono-clonal screening.
4.2 results: tame cultivation by serum-free, rhIL-12 engineering cell can growth go down to posterity and secrete rhIL-12 albumen in CD CHO pressurization substratum; Utilize the method for cloning screening to obtain altogether 10 clones, called after C3.14.1-C3.14.10, amount and the activity of final definite C3.14.6 clone cell secretion rhIL-12 are all better than C3.14 clone cell, the growth conditions of C3.14.6 clone cell in CD CHO pressurization substratum as shown in Figure 1, be shown in shown in Fig. 2, Fig. 3 by the ability of the growth velocity of cell and generation IL-12 albumen.
5. produce the screening with serum free medium: use respectively containing Serum-free and protein-free medium: CD CHO liquid nutrient medium, containing Sodium.alpha.-ketopropionate (0.8-1.2mM), xanthoglobulin (0.075-0.125mM), Thymine deoxyriboside (0.012-0.020mM), adenosine (0.5-0.9mg/L), guanosine-(0.5-0.9mg/L), cytidine(C (0.5-0.9mg/L), uridine (0.5-0.9mg/L), L-glutaminate (0.4-0.8mg/L), altheine (0.4-0.8mg/L), L-PROLINE (1.5-2.0mg/L), the CHO III A liquid nutrient medium of non-essential amino acid (0.08-0.125mM), CD CHO A liquid nutrient medium, CD CHO liquid nutrient medium, SFM liquid nutrient medium and HyQ SFX-CHO serum free medium are cultivated rhIL-12 engineering cell, inoculating cell density is 1 * 10
5/ ml, 37 ℃, 5%CO
2cultivate, cultivate 7 days, draw cells and supernatant every day, for ELISA, detect the content of rhIL-12 and detect its biologic activity.Result shows, in CHO III A liquid nutrient medium, CD CHO A liquid nutrient medium, CD CHO liquid nutrient medium, SFM liquid nutrient medium and HyQ SFX-CHO serum free medium culture supernatant, all there is IL-12 to produce, while using CD CHO culture medium culturing, rhIL-12 protein content the highest (Fig. 4) in CHO-IL12 cells and supernatant.The biological activity of the rhIL-12 albumen that various culture medium culturing produce is all 5.0 * 10
6about IU/mg, cultivates after 7 days Cell viability all more than 90%, no significant difference.CD CHO substratum is the substratum that chemical composition limits, and does not include the component of albumen, hydrolysate or unknown structure in substratum, and all compositions all have known chemical structure.With serum free medium with without albumen, the substratum of determinant is not compared, owing to not containing the composition of animal-origin, lowered the possibility containing the disadvantageous risks and assumptions of human health, and its culture effect has surpassed ordinary culture medium and the serum free medium that must add serum, therefore in conjunction with the requirement of culture medium cost, downstream purification and suitability for industrialized production, finally select CD CHO substratum as the Serum-free and protein-free medium of rhIL-12 engineering cell.
6. the foundation of stable rhIL-12 engineering cell large scale culturing technique:
6.1 use production to cultivate (preferred value) by serum free medium I
6.1.1 produce and use serum free medium I: be contain that final concentration is respectively the Sodium.alpha.-ketopropionate of 1.0mM, the Thymine deoxyriboside of the xanthoglobulin of 0.1mM, 0.016mM, the guanosine-of the adenosine of 0.7mg/L, 0.7mg/L, the uridine of the cytidine(C of 0.7mg/L, 0.7mg/L, the altheine of the L-glutaminate of 0.6mg/L, 0.6mg/L, the CD CHO liquid nutrient medium of the non-essential amino acid of the L-PROLINE of 1.7mg/L, 0.1mM.
6.1.2 cell recovery and static cultivation: (numbering: WCB-171) of (liquid nitrogen container) recovery rhIL-12 engineering cell from working cardial cell storehouse, putting into 37 ℃ of water-baths constantly rocks, it is melted, open after using afterwards 75% ethanol disinfection, with suction pipe sucking-off cell suspension, inject aseptic centrifuge tube, add 20ml CD CHO substratum, 800rpm, 10min eccentric cell, abandoning supernatant, repeated washing once.Use 25ml dosing CD CHO substratum re-suspended cell and be transferred in 250ml sterile culture flask, using platform to expect blue dyeing, counting total cell count and viable count under microscope, calculating Cell viability, result is 92%, 37 ℃ afterwards, and 5%CO
2cultivate, every 2-3 days goes down to posterity once.
6.1.3 the amplification cultivation of engineering cell: by the rhIL-12 engineering cell in logarithmic phase after aseptic Hanks washs (800rpm, 10min, 25 ℃) twice, with 2 * 10
4the density of/ml is inoculated in and is equipped with in 10 liters of big or small culture bag of 2000ml production by serum free medium I.37 ℃, 5.0%CO
2, CO
2flow velocity: 0.12-0.18L/min, 6-10rpm/6 °, cultivates.The secretion situation of the amplification situation of engineering cell in culture bag and rhIL-12 albumen is as follows:
A. ability of cell proliferation analysis: the time-effect relationship of rhIL-12 engineering cell propagation is observed in cell counting, every day is by the culture bag cell that takes a morsel, count and detect Cell viability, cultured continuously 7 days, result shows, rhIL-12 engineering cell has rate of propagation (Fig. 5) faster under the culture condition of culture bag, and Cell viability is all more than 90%.
B.rhIL-12 cell produces protedogenous ability: the cells and supernatant that takes a morsel in culture bag every day, content and the biologic activity thereof of detection rhIL-12 albumen.Result shows, with the prolongation of incubation time, the content of rhIL-12 albumen increases (Fig. 6) gradually, and its activity is stabilized in (5.0-6.5) * 10 always
6iU/mg left and right.
6.1.4 the serum-free culture of engineering cell in fermentor tank:
6.1.4.1 fermentor tank parameter setting: after using sterile purified water that fermentor tank is rinsed well, add the aseptic PBS solution of 5L, proofread and correct pH electrode, connect various gas ducts, stirring velocity 150rpm, is full of water by jacket layer, after machine operates steadily, 121 ℃, 20min autoclaving.Again proofread and correct pH electrode, PBS solution is pumped, add 5L production serum free medium, 50rpm, 37 ℃, logical pressurized air, move after 24 hours.Proofread and correct dissolved oxygen electrode, take final concentration as (3-5) * 10
5/ ml inoculating cell, adjusts parameter: rotating speed is that 50rpm-120rpm, temperature are 36 ℃-37 ℃, and dissolved oxygen is that 30%-60%, pH value be 6.90-7.20, and temperature adopts PID automatic control mode, gas control employing dissolved oxygen-pH master mode that links.
6.1.4.2 perfusion is cultivated: the variation of monitoring at any time dissolved oxygen, glucose, lactic acid and ammonium ion concentration after inoculating cell, according to the variation of oxygen consumption rate, glucose consumption, lactic acid concn, ammonium ion concentration and rhIL-12 (p70) albumen generation etc., carry out perfusion, flow is 0.5-1.5 tank volume.By the monitoring to culturing process, find, remaining sugar concentration in substratum is larger on the impact of the concentration of IL-12 in culture supernatant, in substratum, remaining sugar concentration is too high and when too low, in culture supernatant, the concentration of IL-12 is all lower, when in substratum, remaining sugar concentration is 10mmol/L left and right, in culture supernatant, the expression amount of IL-12 is the highest (Fig. 7).Along with the reduction of remaining sugar concentration in substratum, the concentration of lactic acid rises (Fig. 8) thereupon, and the concentration of ammonium ion also slightly rises.Therefore, according to remaining sugar concentration and lactic acid concn, adjust the interpolation of perfusion flow, glucose and sodium bicarbonate etc., maintain remaining sugar concentration in fermentor tank more than 10mmol/L, control the concentration of lactic acid below 20mmol/L, to improve the expression amount of rhIL-12 albumen.In culturing process, the variation of rhIL-12 protein concentration, lactic acid concn, ammonium ion concentration and oxygen uptake rate as shown in Figure 9.
6.1.5 the collection of serum-free culture supernatant and preservation: when perfusion is produced by serum free medium I, collect culture supernatant in appropriate containers, 12000g, 4 ℃, centrifugal 30 minutes, centrifugal rear supernatant is packed in 1000ml medium bottle,-20 ℃ of preservations, to be purified, whole culturing process continues 65 days, collects altogether culture supernatant 300L.
6.1.6 the concentration of the rhIL-12 (p70) in serum-free culture supernatant and active detection: use ELISA method to detect the concentration of rhIL-12 (p70) in culture supernatant, the mean concns of rhIL-12 in 300L culture supernatant (p70) is 5.8mg/L; Use Western blot method to identify the balance that in culture supernatant, p40 and p35 express, result shows, in serum-free culture supernatant, p40 and p35 all have expression, and ratio approaches 1: 1 (Figure 10); Detect the activity of IL-12 albumen in culture supernatant simultaneously, take NIBSC standard substance (WHO international standard substance) as contrast, and application NKG cell IFN-γ induces method, measures the activity of rhIL-12 in serum-free culture supernatant, result shows, the rhIL-12 specific activity of different batches is all 4.0 * 10
6more than IU/mg.
6.2 use production to cultivate (lower value) by serum free medium II
6.2.1 produce and use serum free medium II: be contain that final concentration is respectively the Sodium.alpha.-ketopropionate of 0.8mM, the Thymine deoxyriboside of the xanthoglobulin of 0.075mM, 0.012mM, the guanosine-of the adenosine of 0.5mg/L, 0.5mg/L, the uridine of the cytidine(C of 0.5mg/L, 0.5mg/L, the altheine of the L-glutaminate of 0.4mg/L, 0.4mg/L, the CD CHO liquid nutrient medium of the non-essential amino acid of the L-PROLINE of 1.5mg/L, 0.08mM.
6.2.2 cell recovery and static cultivation: the same 6.1.2 of experimental technique.
6.2.3 the amplification cultivation of engineering cell: by the rhIL-12 engineering cell in logarithmic phase after aseptic Hanks washs (800rpm, 10min, 25 ℃) twice, with 2 * 10
4the density of/ml is inoculated in and is equipped with in 10 liters of big or small culture bag of 2000ml production by serum free medium II.37 ℃, 5.0%CO
2, CO
2flow velocity: 0.12-0.18L/min, 6-10rpm/6 °, cultivates.The secretion situation of the amplification situation of engineering cell in culture bag and rhIL-12 albumen is as follows:
A. ability of cell proliferation analysis: the time-effect relationship of rhIL-12 engineering cell propagation is observed in cell counting, every day is by the culture bag cell that takes a morsel, count and detect Cell viability, cultured continuously 7 days, result shows, rhIL-12 engineering cell has rate of propagation (Fig. 5) faster under the culture condition of culture bag, and Cell viability is all more than 90%.
B.rhIL-12 cell produces protedogenous ability: the cells and supernatant that takes a morsel in culture bag every day, content and the biologic activity thereof of detection rhIL-12 albumen.Result shows, with the prolongation of incubation time, the content of rhIL-12 albumen increases (Fig. 6) gradually, and its activity is stabilized in (5.0-6.5) * 10 always
6iU/mg left and right.
6.2.4 the serum-free culture of engineering cell in fermentor tank:
6.2.4.1 fermentor tank parameter setting: after using sterile purified water that fermentor tank is rinsed well, add the aseptic PBS solution of 5L, proofread and correct pH electrode, connect various gas ducts, stirring velocity 150rpm, is full of water by jacket layer, after machine operates steadily, 121 ℃, 20min autoclaving.Again proofread and correct pH electrode, PBS solution is pumped, add 5L to produce and use serum free medium II, 50rpm, 37 ℃, logical pressurized air, move after 24 hours.Proofread and correct dissolved oxygen electrode, take final concentration as (3-5) * 10
5/ ml inoculating cell, adjusts parameter: rotating speed is that 50rpm-120rpm, temperature are 36 ℃-37 ℃, and dissolved oxygen is that 30%-60%, pH value be 6.90-7.20, and temperature adopts PID automatic control mode, gas control employing dissolved oxygen-pH master mode that links.
6.2.4.2 perfusion is cultivated: the variation of monitoring at any time dissolved oxygen, glucose, lactic acid and ammonium ion concentration after inoculating cell, according to the variation of oxygen consumption rate, glucose consumption, lactic acid concn, ammonium ion concentration and rhIL-12 (p70) albumen generation etc., carry out perfusion, flow is 0.5-1.5 tank volume.By the monitoring to culturing process, find, remaining sugar concentration in substratum is larger on the impact of the concentration of IL-12 in culture supernatant, in substratum, remaining sugar concentration is too high and when too low, in culture supernatant, the concentration of IL-12 is all lower, when in substratum, remaining sugar concentration is 10mmol/L left and right, in culture supernatant, the expression amount of IL-12 is the highest (Fig. 7).Along with the reduction of remaining sugar concentration in substratum, the concentration of lactic acid rises (Fig. 8) thereupon, and the concentration of ammonium ion also slightly rises.Therefore, according to remaining sugar concentration and lactic acid concn, adjust the interpolation of perfusion flow, glucose and sodium bicarbonate etc., maintain remaining sugar concentration in fermentor tank more than 10mmol/L, control the concentration of lactic acid below 20mmol/L, to improve the expression amount of rhIL-12 albumen.In culturing process, the variation of rhIL-12 protein concentration, lactic acid concn, ammonium ion concentration and oxygen uptake rate as shown in Figure 9.
6.2.5 the collection of serum-free culture supernatant and preservation: when perfusion is produced by Serum-free and protein-free medium II, collect culture supernatant in appropriate containers, 12000g, 4 ℃, centrifugal 30 minutes, centrifugal rear supernatant is packed in 1000ml medium bottle,-20 ℃ of preservations, to be purified, whole culturing process continues 65 days, collects altogether culture supernatant 310L.
6.2.6 the concentration of the rhIL-12 (p70) in serum-free culture supernatant and active detection: use ELISA method to detect the concentration of rhIL-12 (p70) in culture supernatant, the mean concns of rhIL-12 in 300L culture supernatant (p70) is 5.75mg/L; Use Western blot method to identify the balance that in culture supernatant, p40 and p35 express, result shows, in serum-free culture supernatant, p40 and p35 all have expression, and ratio approaches 1: 1 (Figure 10); Detect the activity of IL-12 albumen in culture supernatant simultaneously, take NIBSC standard substance (WHO international standard substance) as contrast, and application NKG cell IFN-γ induces method, measures the activity of rhIL-12 in serum-free culture supernatant, result shows, the rhIL-12 specific activity of different batches is all 4.0 * 10
6more than IU/mg.
6.3 use production to cultivate (higher limit) by serum free medium III
6.3.1 produce and use serum free medium III: be contain that final concentration is respectively the Sodium.alpha.-ketopropionate of 1.2mM, the Thymine deoxyriboside of the xanthoglobulin of 0.125mM, 0.020mM, the guanosine-of the adenosine of 0.9mg/L, 0.9mg/L, the uridine of the cytidine(C of 0.9mg/L, 0.9mg/L, the altheine of the L-glutaminate of 0.8mg/L, 0.8mg/L, the CD CHO liquid nutrient medium of the non-essential amino acid of the L-PROLINE of 2.0mg/L, 0.125mM.
6.3.2 cell recovery and static cultivation: the same 6.1.2 of experimental technique.
6.3.3 the amplification cultivation of engineering cell: by the rhIL-12 engineering cell in logarithmic phase after aseptic Hanks washs (800rpm, 10min, 25 ℃) twice, with 2 * 10
4the density of/ml is inoculated in and is equipped with in 10 liters of big or small culture bag of 2000ml production by serum free medium III.37 ℃, 5.0%CO
2, CO
2flow velocity: 0.12-0.18L/min, 6-10rpm/6 °, cultivates.The secretion situation of the amplification situation of engineering cell in culture bag and rhIL-12 albumen is as follows:
A. ability of cell proliferation analysis: the time-effect relationship of rhIL-12 engineering cell propagation is observed in cell counting, every day is by the culture bag cell that takes a morsel, count and detect Cell viability, cultured continuously 7 days, result shows, rhIL-12 engineering cell has rate of propagation (Fig. 5) faster under the culture condition of culture bag, and Cell viability is all more than 90%.
B.rhIL-12 cell produces protedogenous ability: the cells and supernatant that takes a morsel in culture bag every day, content and the biologic activity thereof of detection rhIL-12 albumen.Result shows, with the prolongation of incubation time, the content of rhIL-12 albumen increases (Fig. 6) gradually, and its activity is stabilized in (5.0-6.5) * 10 always
6iU/mg left and right.
6.3.4 the serum-free culture of engineering cell in fermentor tank:
6.3.4.1 fermentor tank parameter setting: after using sterile purified water that fermentor tank is rinsed well, add the aseptic PBS solution of 5L, proofread and correct pH electrode, connect various gas ducts, stirring velocity 150rpm, is full of water by jacket layer, after machine operates steadily, 121 ℃, 20min autoclaving.Again proofread and correct pH electrode, PBS solution is pumped, add 5L to produce and use serum free medium III, 50rpm, 37 ℃, logical pressurized air, move after 24 hours.Proofread and correct dissolved oxygen electrode, take final concentration as (3-5) * 10
5/ ml inoculating cell, adjusts parameter: rotating speed is that 50rpm-120rpm, temperature are 36 ℃-37 ℃, and dissolved oxygen is that 30%-60%, pH value be 6.90-7.20, and temperature adopts PID automatic control mode, gas control employing dissolved oxygen-pH master mode that links.
6.3.4.2 perfusion is cultivated: the variation of monitoring at any time dissolved oxygen, glucose, lactic acid and ammonium ion concentration after inoculating cell, according to the variation of oxygen consumption rate, glucose consumption, lactic acid concn, ammonium ion concentration and rhIL-12 (p70) albumen generation etc., carry out perfusion, flow is 0.5-1.5 tank volume.By the monitoring to culturing process, find, remaining sugar concentration in substratum is larger on the impact of the concentration of IL-12 in culture supernatant, in substratum, remaining sugar concentration is too high and when too low, in culture supernatant, the concentration of IL-12 is all lower, when in substratum, remaining sugar concentration is 10mmol/L left and right, in culture supernatant, the expression amount of IL-12 is the highest (Fig. 7).Along with the reduction of remaining sugar concentration in substratum, the concentration of lactic acid rises (Fig. 8) thereupon, and the concentration of ammonium ion also slightly rises.Therefore, according to remaining sugar concentration and lactic acid concn, adjust the interpolation of perfusion flow, glucose and sodium bicarbonate etc., maintain remaining sugar concentration in fermentor tank more than 10mmol/L, control the concentration of lactic acid below 20mmol/L, to improve the expression amount of rhIL-12 albumen.In culturing process, the variation of rhIL-12 protein concentration, lactic acid concn, ammonium ion concentration and oxygen uptake rate as shown in Figure 9.
6.3.5 the collection of serum-free culture supernatant and preservation: when perfusion is produced by Serum-free and protein-free medium II, collect culture supernatant in appropriate containers, 12000g, 4 ℃, centrifugal 30 minutes, centrifugal rear supernatant is packed in 1000ml medium bottle,-20 ℃ of preservations, to be purified, whole culturing process continues 64 days, collects altogether culture supernatant 295L.
6.3.6 the concentration of the rhIL-12 (p70) in serum-free culture supernatant and active detection: use ELISA method to detect the concentration of rhIL-12 (p70) in culture supernatant, the mean concns of rhIL-12 in 300L culture supernatant (p70) is 5.70mg/L; Use Western blot method to identify the balance that in culture supernatant, p40 and p35 express, result shows, in serum-free culture supernatant, p40 and p35 all have expression, and ratio approaches 1: 1 (Figure 10); Detect the activity of IL-12 albumen in culture supernatant simultaneously, take NIBSC standard substance (WHO international standard substance) as contrast, and application NKG cell IFN-γ induces method, measures the activity of rhIL-12 in serum-free culture supernatant, result shows, the rhIL-12 specific activity of different batches is all 4.0 * 10
6more than IU/mg.
Use the technical process that the present invention sets up to cultivate rhIL-12 engineering cell, more than in culture supernatant, the content of rhIL-12 albumen can reach 5mg/L, protein-active is 4.0 * 10
6more than IU/mg, the content of rhIL-12, activity all can meet the requirement of downstream purification and clinical application, particularly the application of Serum-free and protein-free medium can reduce the risk of the step of purifying and cost, reduction clinical application, for theoretical investigation and the clinical application of IL-12 albumen provides technical guarantee.
Claims (10)
1. the extensive serum-free culture method of rhIL-12 engineering cell, comprises the steps:
RhIL-12 engineering cell in logarithmic phase is inoculated in Serum-free and protein-free medium and is cultivated, described substratum is to contain the Sodium.alpha.-ketopropionate that final concentration is respectively 0.8-1.2mM, the xanthoglobulin of 0.075-0.125mM, the Thymine deoxyriboside of 0.012-0.020mM, the adenosine of 0.5-0.9mg/L, the guanosine-of 0.5-0.9mg/L, the cytidine(C of 0.5-0.9mg/L, the uridine of 0.5-0.9mg/L, the L-glutaminate of 0.4-0.8mg/L, the altheine of 0.4-0.8mg/L, the L-PROLINE of 1.5-2.0mg/L, the CD CHO liquid nutrient medium of the non-essential amino acid of 0.08-0.125mM.
2. the extensive serum-free culture method of rhIL-12 engineering cell according to claim 1, wherein said cultivation is carried out in fermentor tank.
3. the extensive serum-free culture method of rhIL-12 engineering cell according to claim 1 and 2, the remaining sugar concentration wherein maintaining in described substratum is 10mmol/L.
4. the extensive serum-free culture method of rhIL-12 engineering cell according to claim 1 and 2, wherein maintains remaining sugar concentration in described substratum more than 10mmol/L, and controls lactic acid concn below 20mmol/L.
5. the extensive serum-free culture method of rhIL-12 engineering cell according to claim 1 and 2, wherein said rhIL-12 engineering cell obtains by pressurization Screening of Media, described pressurization substratum is to contain the Sodium.alpha.-ketopropionate that final concentration is respectively 1.0mM, the xanthoglobulin of 0.1mM, the Thymine deoxyriboside of 0.016mM, the adenosine of 0.7mg/L, the guanosine-of 0.7mg/L, the cytidine(C of 0.7mg/L, the uridine of 0.7mg/L, the L-glutaminate of 0.6mg/L, the altheine of 0.6mg/L, the L-PROLINE of 1.7mg/L, the non-essential amino acid of 0.1mM, the methionine sulfoxide of 500 μ M, the CD CHO liquid nutrient medium of the G418 of 800mg/L.
6. the extensive serum-free culture method of rhIL-12 engineering cell according to claim 1 and 2, the step of cultivating comprising perfusion.
7. the extensive serum-free culture method of rhIL-12 engineering cell according to claim 1 and 2, comprising collecting and the step of preserving the rhIL-12 in described substratum supernatant.
8. the extensive serum-free culture method of rhIL-12 engineering cell according to claim 1 and 2, the rhIL-12 content wherein obtaining by described method is more than 5mg/L.
9. the extensive serum-free culture method of rhIL-12 engineering cell according to claim 1 and 2, in the rhIL-12 wherein obtaining by described method, the ratio of p40 and p35 is 1: 1.
10. a rhIL-12 engineering cell Serum-free and protein-free medium, described substratum is to contain the Sodium.alpha.-ketopropionate that final concentration is respectively 0.8-1.2mM, the xanthoglobulin of 0.075-0.125mM, the Thymine deoxyriboside of 0.012-0.020mM, the adenosine of 0.5-0.9mg/L, the guanosine-of 0.5-0.9mg/L, the cytidine(C of 0.5-0.9mg/L, the uridine of 0.5-0.9mg/L, the L-glutaminate of 0.4-0.8mg/L, the altheine of 0.4-0.8mg/L, the L-PROLINE of 1.5-2.0mg/L, the CD CHO liquid nutrient medium of the non-essential amino acid of 0.08-0.125mM.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110208009.8A CN102268407B (en) | 2011-07-20 | 2011-07-20 | Large-scale serum-free culture method for rhIL-12 engineering cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201110208009.8A CN102268407B (en) | 2011-07-20 | 2011-07-20 | Large-scale serum-free culture method for rhIL-12 engineering cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102268407A CN102268407A (en) | 2011-12-07 |
| CN102268407B true CN102268407B (en) | 2014-04-02 |
Family
ID=45050871
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201110208009.8A Active CN102268407B (en) | 2011-07-20 | 2011-07-20 | Large-scale serum-free culture method for rhIL-12 engineering cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102268407B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320390B (en) * | 2013-06-17 | 2015-03-25 | 齐鲁制药有限公司 | Large-scale mammal engineering cell cultural method |
| CN109207424A (en) * | 2018-10-24 | 2019-01-15 | 河南省肿瘤医院 | A kind of cultural method of immunocyte |
| CN114591885B (en) * | 2022-03-05 | 2024-02-23 | 河南普华基因科技有限公司 | LLC-PK1Sa cell domestication method suitable for serum-free suspension culture and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1467292A (en) * | 2003-05-27 | 2004-01-14 | 中国科学技术大学 | Highly effective expressing method of interleukin-12 |
-
2011
- 2011-07-20 CN CN201110208009.8A patent/CN102268407B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1467292A (en) * | 2003-05-27 | 2004-01-14 | 中国科学技术大学 | Highly effective expressing method of interleukin-12 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102268407A (en) | 2011-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108004202B (en) | Culture solution for serum-free suspension culture of 293T cells | |
| CN101864396A (en) | Method for inducing megakaryoblast and megakaryocyte in vitro | |
| CN102517251A (en) | Mesenchymal stem cells, as well as preparation method and application thereof | |
| CN102443565B (en) | Medium suitable for cultivating CHO cell and cultivation technology thereof | |
| CN102100910A (en) | Method for producing viral vaccines | |
| CN110462054A (en) | Culture medium is perfused | |
| CN102268407B (en) | Large-scale serum-free culture method for rhIL-12 engineering cells | |
| CN101831403B (en) | Method for amplifying mesenchymal stem cells of human umbilical cord and placenta in vitro | |
| CN103160478B (en) | Optimized technological method for amplifying recombinant adenovirus by using bioreactor | |
| CN103320390A (en) | Large-scale mammal engineering cell cultural method | |
| CN102988972B (en) | Method for producing porcine parvovirus inactivated vaccine by using torrent bioreactor | |
| CN107523555A (en) | The method for obtaining virus | |
| Okonkowski et al. | Cholesterol delivery to NS0 cells: challenges and solutions in disposable linear low-density polyethylene-based bioreactors | |
| CN102719397B (en) | Mesenchymal stem cell and application thereof in resisting HIV-1 (human immunodeficiency virus) | |
| CN104789594A (en) | Method for constructing CHO cell strain for stably and efficiently expressing human serum albumin and interleukin II fusion protein | |
| CN103160458A (en) | Low-serum medium suitable for growth of Vero cells | |
| CN112094814B (en) | Method for preparing adenovirus vector vaccine by perfusion culture process | |
| US9932562B2 (en) | Drain down and re-feed of microcarrier bioreactor | |
| CN103484426B (en) | Non-animal source low-protein culture medium | |
| CN109576212B (en) | Culture method of seed cells in high-density inoculation culture and application thereof | |
| CN102776260A (en) | Method for effectively expressing recombinant human coagulation factor VIII | |
| CN104593334A (en) | Method for producing ring II type DBN-SX07 strain virus for pigs by seroculturing PK-15 cells | |
| CN105018423A (en) | CIK cell culturing method | |
| CN104630159A (en) | Method for producing hog cholera C-strain virus by culturing ST Cells in low serum | |
| CN109628407A (en) | The method of high density long-term cultivation expression anti-vegf Humanized monoclonal antibodies recombinaant CHO cell |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |