CN101603026A - Be suitable for the animal origin-free low-protein culture medium of zooblast products production - Google Patents
Be suitable for the animal origin-free low-protein culture medium of zooblast products production Download PDFInfo
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Abstract
The present invention is suitable for the animal origin-free low-protein culture medium of zooblast products production, comprise 24 kinds of basal metabolism nutrition, 11 kinds of VITAMIN, 3 kinds of Transferrins,iron complexes surrogate compounds, 5 kinds of lipoid substances, 2 kinds of nucleic acid compounds, 4 kinds of hormones and somatomedin, 3 kinds of antioxidants, a kind of anti-shearing protective material, a kind of pH indicator, 2 kinds of pH buffer reagents, 9 kinds of other inorganic salt, adopt the soybean hydrolyzate to substitute animal derived components, adopt the composition of ferrous sulfate, iron nitrate and EDTA2Na to substitute Transferrins,iron complexes; Said components is dissolved in three distilled water, can makes described substratum; Its positively effect is: do not contain animal derived components, total protein content is lower than 10 mg/litre, help the separation and purification of product, be suitable for recombinant protein class drug manufacture, support the normal growth and the cultivation of going down to posterity for a long time of zooblast, need not to adapt to and can use, preparation is convenient, is suitable for the scale operation of zooblast product.
Description
[technical field]
The present invention relates to the substratum research and development technology field of modern biotechnology, specifically, is a kind of animal origin-free low-protein culture medium that is suitable for recombinant protein medicine products productions such as animal cell large-scale cultivation and antibody, fusion rotein.
[technical background]
The animal cell culture technology is one of advanced subject in the current bioengineering field, is the attention that fundamental research or production practice aspect all obtain biotechnology circle.Since 2000, in all innovation biotech drugs of FDA approval,,, and reached 55 kinds, account for more than 70% with the medicine of mammalian cell expression or production with have only 6 kinds of escherichia coli expression with have only 2 kinds of yeast expression.In 10 biotech drugs that year global marketing in June volume is the highest of in June, 2003～2004, the product of mammalian cell expression accounts for 8, and annual sales amount to surpass 2,000,000,000 dollars preceding 6 biotech drug all be the product of mammalian cell expression.Global biotech drug sales volume surpassed 50,000,000,000 dollars in 2005, and the mammalian cell expression product accounts for 70%.To the year ends 2007, only the sales volume of monoclonal antibody drug just reaches 26,000,000,000 dollars, estimates will reach 49,000,000,000 dollars to its sales volume in 2013.Mass-producing cultivation and expression of recombinant proteins production that application serum-free culture technology is carried out zooblast have become a kind of trend.
Along with mammalian cell is cultivated the expansion and the bio-pharmaceutical growth of requirement of scale, become the important topic in cell engineering field based on the development of the serum free medium of cell and product performance.In the Application Areas of biological products such as production of vaccine, monoclonal antibody and various biological activity proteins, optimize the composition of serum free medium, making engineering cell keep higher viable cell density and long incubation time in the culture environment that helps most its growth and the expression of purpose product, is the effective ways that reduce production costs.
Compare with traditional blood serum medium that has, serum free medium has many advantages: the first, and do not have batch difference, do not have the influence of failing to understand the serum component pair cell; The second, avoided the toxic action of exogenous pollution and pair cell in the serum; The 3rd, make product be easy to purifying, improve the rate of recovery; The 4th, definite ingredients helps studying physiological status of cells, can and optimize the substratum that can support its high-density culture and product high level expression according to different cell strain designs.In addition, the serum-free culture technology also is one of gordian technique of carrying out fundamental researchs such as cell growth, propagation, differentiation and gene expression regulation.
The exploitation of serum free medium and optimization mainly contain following two developing direction: the one, do not add the composition of any animal-origin; The 2nd, the protein content of reduction substratum.In view of the above, serum free medium can be divided into following three kinds:
(1) traditional serum free medium, promptly use the component of all kinds of alternative serum functions formulated, as add components such as bovine serum albumin (BSA), Transferrins,iron complexes, Regular Insulin, the mixed lipids of from serum, extracting and protolysate, be characterized in the protein content height, contain a large amount of protein for animal.
(2) substratum of animal origin-free composition is promptly dispeled the animal derived components in the substratum.Owing to may contain the danger of potential PrPC and virus pollution in the animal derived components, therefore, must the careful animal derived components of using in substratum, can guarantee that so just the recombinant protein of producing is safe and reliable.
(3) low albumen or protein-free medium promptly reduce or dispel protein component in the substratum.Reduce the protein content in the substratum, can reduce the purification procedures of product, improve the rate of recovery and the quality of target protein, the consistence between guaranteeing batch, thus realize the high-level efficiency and the economy of whole process of production.
In sum, the exploitation of Methods of Serum-Free Medium for Animal Cells and optimization are that direction towards " animal origin-free, no albumen " develops.
Through the development of decades, mammalian cell (as hybridoma, Chinese hamster ovary cell (CHO), NSO etc.) serum free medium technology has obtained gratifying progress:
People such as Darfler have developed the CITTL serum free medium, with DMEM/F12 (1: 1) is basic medium, the catalase of 5mg/L, the Regular Insulin of 1.5mg/L, Transferrins,iron complexes, 2nmol/L testosterone, the two inferior oleoyl phosphatidyl cholines of 0.5mg/L and the β-Phosphoric acid glycerol esters of 1.5mg/L of 1.5～3.0mg/L have been added, can support the cloning growth of sf9 cell, also can be used for the cultivation of hybridoma.
People such as Murakami have developed serum free medium, with DMEM/F12 (1: 1) is basic medium, materials such as 5mg/L Regular Insulin, 2～35mg/L Transferrins,iron complexes, 20 μ mol/L thanomins and 1nmol/L Sodium Selenite have been added, the DMEM/F12-ITES prescription that Here it is now is widely used.
Kover and Frank have developed serum free medium, with RPMI1640 is basic medium, and add 10mg/L Regular Insulin, 5mg/L Transferrins,iron complexes, 20 μ mol/L thanomins, 5mg/L linolic acid, 1g/L bovine serum albumin, 3mg/L xitix, 2 μ g hydrocortisones and 12 kinds of trace elements, can support the growth of several cells.
United States Patent (USP) U.S.P.4927762 discloses a kind of substratum that contains antioxidant, having set forth cell can grow in reductant better, point out that simultaneously the interpolation of materials such as N-acetylcystein, thiolactic acid, mercaptoethanol can be played oxidation resistant effect.
United States Patent (USP) U.S.P.4767704 mainly studies with regard to the interpolation of trace element, by interpolation to materials such as elements such as copper, iron, magnesium, zinc, silicon, manganese, vanadium, nickel, tin, aluminium, calcium, bromine, chlorine, iodine, selenium, germanium and progesterone, putrescine, oleic acid, pyrimidines, developed a kind of protein-free medium, made people recognize the importance of trace element.
People such as Jinyou Zhang are with Fe-Na
2SeO
4The mixture replacing Transferrins,iron complexes, the gonad cell of developing (CHO) protein-free medium can be supported higher viable cell density.But Fe-Na
2SeO
4The preparation of mixture is very complicated, and the condition harshness is not suitable for large-scale production.
At present, with Gibico, Hyclone, JRH etc. also come out one after another for commercial serum-free, the protein-free medium of representative.In existing serum free medium, some can't realize high-density culture process; Though some sustenticular cell growth well may cause the cell expression product ability to reduce, even forfeiture; In addition, the prescription of commercial serum free medium often belongs to trade secret, and the component complexity, costs an arm and a leg, and both has been unfavorable for the research of specific cells strain culture technique, also can't be directly used in research, exploitation and the optimization of cell cultivation process.
[summary of the invention]
The objective of the invention is to overcome the deficiencies in the prior art, a kind of animal origin-free low-protein culture medium that is suitable for the zooblast products production is provided, animal cell large-scale is cultivated and the needs of industrial production recombinant protein medicine product to adapt to.
For achieving the above object, the technical scheme taked of the present invention is:
A kind of animal origin-free low-protein culture medium that is suitable for the zooblast products production, it is characterized in that, the alternative animal derived components of employing soybean hydrolyzate (as, bovine serum albumin etc.), adopt the composition of ferrous sulfate, iron nitrate and EDTA2Na to substitute Transferrins,iron complexes, its composition comprises basal metabolism nutrition class, vitamins, Transferrins,iron complexes surrogate, lipid, nucleic acid class, hormone and somatomedin class, antioxidant, anti-shearing protective material, pH indicator, pH buffer reagent and other inorganic salts;
---described basal metabolism nutrition class comprises: glucose, soybean hydrolyzate, Sodium.alpha.-ketopropionate, glutamine, L-Ala, arginine, l-asparagine, aspartic acid, halfcystine, Gelucystine, L-glutamic acid, glycine, Histidine, Isoleucine, leucine, Methionin, methionine(Met), phenylalanine, proline(Pro), Serine, Threonine, tryptophane, tyrosine, Xie Ansuan;
---described vitamins comprises: vitamin H, VITAMIN, choline chloride 60, folic acid, inositol, niacinamide, calcium pantothenate, vitamin B6, riboflavin, VITMAIN B1, Thioctic Acid;
---described Transferrins,iron complexes surrogate class comprises: ferrous sulfate, iron nitrate, EDTA2Na;
---described lipid comprises: thanomin, tween 80, Oils,glyceridic,cod-liver, cholesterol, linolic acid;
---described nucleic acid class comprises: thymidine, xanthoglobulin;
---described hormone and somatomedin class comprise: progesterone, hydrocortisone, Recombulin, putrescine;
---described antioxidant comprises: Sodium Selenite, tocopherol, taurine;
---described anti-shearing protective material is blocked polyethers F68;
---described pH indicator is phenol red;
---described pH buffer reagent comprises sodium bicarbonate, Hepes;
---described other inorganic salts comprise: calcium chloride, magnesium chloride, sal epsom, Repone K, SODIUM PHOSPHATE, MONOBASIC, sodium-chlor, Sodium phosphate dibasic, zinc sulfate, copper sulfate.
A kind of animal origin-free low-protein culture medium that is suitable for the zooblast products production, the content of its various compositions are (mg/litre):
Glucose (
D-Glucose) 2000～7000
Soybean hydrolyzate (Lucratone Soy) 500～1000
Sodium.alpha.-ketopropionate (Sodium Pyruvate) 55～220
Glutamine (
L-Glutamine) 200～900
L-Ala (
L-Alanine) 4～15
Arginine (
L-ArginineHCl) 100～300
L-asparagine (
L-AsparagineH
2O) 6～15
Aspartic acid (
L-Aspartic Acid) 6～15
Halfcystine (
L-CysteineH
2O) 15～50
Gelucystine (
L-Cystine2HCl) 30～65
L-glutamic acid (
L-Glutamic Acid) 5～20
Glycine (Glycine) 15～40
Histidine (
L-HistidineHClH
2O) 15～40
Isoleucine (
L-Isoleucine) 50～100
Leucine (
L-Leucine) 50～150
Methionin (
L-LysineHCl) 80～185
Methionine(Met) (
L-Methionine) 15～35
Phenylalanine (
L-Phenyalanine) 30～70
Proline(Pro) (
L-Proline) 10～34.5
Serine (
L-Serine) 20～52.5
Threonine (
L-Threonine) 50～107
Tryptophane (
L-Tryptophan) 9～18
Tyrosine (
L-Tyrosine2Na2H
2O) 55～120
Xie Ansuan (
L-Valine) 50～120
Vitamin H (Biotin) 0.0035～0.0105
Vitamin B12 (Vitamin B12) 0.50～2.50
Choline chloride 60 (Choline Chloride) 8.50～30
Folic acid (Folic acid) 2.50～8
Inositol (
I-Nositol) 12～30
Niacinamide (Niacinamide) 2～7
Calcium pantothenate (
D-CaPantothenate) 2～8
Vitamin B6 (PyridoxineHCl) 2～8
Riboflavin (Riboflavin) 0.2～0.7
VITMAIN B1 (ThiamineHCl) 2～8
Thioctic Acid (Lipoic acid) 0.08～0.50
Ferrous sulfate (FeSO
47H
2O) 0.417～5
Iron nitrate (Fe (NO
3)
39H
2O) 0.05～0.8
EDTA·2Na 2～10
Thanomin (Ethanolamine) 3～10
Tween 80 (Tween 80) 0.24～0.5
Oils,glyceridic,cod-liver (Cod liver oil) 0.1～0.2
Cholesterol (Cholesterol) 0.045～0.1
Linolic acid (Linoleic acid) 0.042～0.084
Thymidine (Thymidine) 0.4～2.5
Xanthoglobulin (Hypoxanthine) 2～20
Progesterone (Progesterone) 0.003～0.012
Hydrocortisone (HydrocortisoneHCl) 0.036～0.072
Recombulin (Insulin) 3～8
Putrescine (Sodium PutrescineHCl) 0.08～20
Sodium Selenite (Sodium Selenate) 0.02～0.08
Tocopherol (α-Tocohcrol acetate) 1～4
Taurine (Taurin) 20～50
Blocked polyethers F68 (Pluronic-F68) 500～2000
Phenol red (Phenol Red) 8.1
Sodium bicarbonate (NaHCO
3) 2450
Hepes 2000～5000
Calcium chloride (CaCl
2) 110～150
Magnesium chloride (MgCl
2) 40～75
Sal epsom (MgSO
4) 45～65
Repone K (KCl) 310～400
SODIUM PHOSPHATE, MONOBASIC (NaH
2PO
4H
2O) 60～120
Sodium-chlor (NaCl) 6995.5
Sodium phosphate dibasic (Na
2HPO
4) 55～120
Zinc sulfate (ZnSO
47H
2O) 0.432～1.25
Copper sulfate (CuSO
45H
2O) 0.0013～0.0065.
Positively effect of the present invention is:
(1) do not contain animal derived components, total protein content is lower than 10 mg/litre, helps the separation and purification of product, is suitable for the recombinant protein class medicament production production of animal cell expression, and is safe and reliable.
(2) normal growth, expression of recombinant proteins and the cultivation of going down to posterity for a long time of support zooblast need not to adapt to and can use, and its highest viable cell density and recombinant protein output are all above known commercial substratum.
(3) composition is simple, and preparation is convenient, is suitable for the scale operation of zooblast product.
[description of drawings]
Accompanying drawing is that Chinese hamster ovary celI is criticized incubation growth in animal origin-free low-protein culture medium of the present invention and product is expressed the coordinate diagram of curve.
[embodiment]
Below the present invention is suitable for the animal origin-free low-protein culture medium of zooblast products production embodiment be specifically described, but the invention is not restricted to following embodiment.
A kind of animal origin-free low-protein culture medium that is suitable for the zooblast products production; comprise 24 kinds of basal metabolism nutrition, 11 kinds of VITAMIN, 3 kinds of Transferrins,iron complexes surrogate compounds, 5 kinds of lipoid substances, 2 kinds of nucleic acid compounds, 4 kinds of hormones and somatomedin, 3 kinds of antioxidants, a kind of anti-shearing protective material, a kind of pH indicator, 2 kinds of pH buffer reagents, 9 kinds of other inorganic salt, the content of various compositions is (mg/litre):
Glucose (
D-Glucose) 4500
Soybean hydrolyzate (Lucratone Soy) 1000
Sodium.alpha.-ketopropionate (Sodium Pyruvate) 55
Glutamine (
L-Glutamine) 584.6
L-Ala (
L-Alanine) 5
Arginine (
L-ArginineHCl) 155
L-asparagine (
L-AsparagineH
2O) 7.5
Aspartic acid (
L-Aspartic Acid) 6.65
Halfcystine (
L-CysteineH
2O) 17.56
Gelucystine (
L-Cystine2HCl) 31.29
L-glutamic acid (
L-Glutamic Acid) 7.35
Glycine (Glycine) 18.75
Histidine (
L-HistidineHClH
2O) 18.75
Isoleucine (
L-Isoleucine) 54.47
Leucine (
L-Leucine) 59.05
Methionin (
L-LysineHCl) 91.25
Methionine(Met) (
L-Methionine) 17.24
Phenylalanine (
L-Phenyalanine) 35.48
Proline(Pro) (
L-Proline) 17.25
Serine (
L-Serine) 26.25
Threonine (
L-Threonine) 53.45
Tryptophane (
L-Tryptophan) 9.02
Tyrosine (
L-Tyrosine2Na2H
2O) 55.79
Xie Ansuan (
L-Valine) 52.85
Vitamin H (Biotin) 0.0105
Vitamin B12 (Vitamin B12) 2.04
Choline chloride 60 (Choline Chloride) 26.94
Folic acid (Folic acid) 7.95
Inositol (
I-Nositol) 12.6
Niacinamide (Niacinamide) 6.06
Calcium pantothenate (
D-CaPantothenate) 6.72
Vitamin B6 (PyridoxineHCl) 6.093
Riboflavin (Riboflavin) 0.657
VITMAIN B1 (ThiamineHCl) 6.51
Thioctic Acid (Lipoic acid) 0.305
Ferrous sulfate (FeSO
47H
2O) 1.217
Iron nitrate (Fe (NO
3)
39H
2O) 0.65
EDTA·2Na 5
Thanomin (Ethanolamine) 9
Tween 80 (Tween 80) 0.24
Oils,glyceridic,cod-liver (Cod liver oil) 0.1
Cholesterol (Cholesterol) 0.045
Linolic acid (Linoleic acid) 0.042
Thymidine (Thymidine) 2.365
Xanthoglobulin (Hypoxanthine) 10
Progesterone (Progesterone) 0.006
Hydrocortisone (HydrocortisoneHCl) 0.036
Regular Insulin (Insulin) 8
Putrescine (Sodium PutrescineHCl) 0.081
Sodium Selenite (Sodium Selenate) 0.0519
Progesterone (α-Tocohcrol acetate) 2
Taurine (Taurin) 30
Blocked polyethers F68 (Pluronic-F68) 1000
Phenol red (Phenol Red) 8.1
Sodium bicarbonate (NaHCO
3) 2450
Hepes 3500
Calcium chloride (CaCl
2) 116.6
Magnesium chloride (MgCl
2) 40.4
Sal epsom (MgSO
4) 48.84
Repone K (KCl) 311.8
SODIUM PHOSPHATE, MONOBASIC (NaH
2PO
4H
2O) 120
Sodium-chlor (NaCl) 6995.5
Sodium phosphate dibasic (Na
2HPO
4) 110
Copper sulfate (CuSO
45H
2O) 0.006
Zinc sulfate (ZnSO
47H
2O) 0.432.
Said components is dissolved in three distilled water, can be mixed with the animal origin-free low-protein culture medium that is suitable for the zooblast products production.
Use animal origin-free low-protein culture medium of the present invention, the Chinese hamster ovary cell (CHO) of expressing anti-CD20 chimeric mAb is inoculated in 2 liters of bio-reactors, inoculum density is 2.39 * 10
5Cells/ml, cytoactive is 98.45% during inoculation; Cell carries out 155 hours batch cultivation in these 2 liters of reactors, the average specific growth velocity of logarithmic phase is 0.652 day
-1, maximum viable cell density reaches 3.75 * 10
6Cells/ml, 15.7 times of cell amplifications, maximum monoclonal antibody concentration is 203 mg/litre, viable cell density is 13.41 * 10 to the integrated value of time
9The cell sky, the antibody production of logarithmic phase unit cell every day reaches 15.88 milligrams/10
9The cell sky.
Accompanying drawing is that Chinese hamster ovary celI is criticized incubation growth in animal origin-free low-protein culture medium of the present invention and product is expressed the coordinate diagram of curve, attached middle school,
◆ the viable cell curve;
■ dead cell curve;
▲ total cell curve;
product curve.
Can learn that from the curve of accompanying drawing the present invention is suitable for the animal origin-free low-protein culture medium normal growth and the product expression of sustenticular cell well of zooblast products production, its highest viable cell density and production concentration are all above known commercial substratum.
Claims (2)
1, a kind of animal origin-free low-protein culture medium that is suitable for the zooblast products production, it is characterized in that, adopt the soybean hydrolyzate to substitute animal derived components, adopt the composition of ferrous sulfate, iron nitrate and EDTA2Na to substitute Transferrins,iron complexes, its composition comprises basal metabolism nutrition class, vitamins, Transferrins,iron complexes surrogate, lipid, nucleic acid class, hormone and somatomedin class, antioxidant, anti-shearing protective material, pH indicator, pH buffer reagent and other inorganic salts;
---described basal metabolism nutrition class comprises: glucose, soybean hydrolyzate, Sodium.alpha.-ketopropionate, glutamine, L-Ala, arginine, l-asparagine, aspartic acid, halfcystine, Gelucystine, L-glutamic acid, glycine, Histidine, Isoleucine, leucine, Methionin, methionine(Met), phenylalanine, proline(Pro), Serine, Threonine, tryptophane, tyrosine, Xie Ansuan;
---described vitamins comprises: vitamin H, VITAMIN, choline chloride 60, folic acid, inositol, niacinamide, calcium pantothenate, vitamin B6, riboflavin, VITMAIN B1, Thioctic Acid;
---described Transferrins,iron complexes surrogate class comprises: ferrous sulfate, iron nitrate, EDTA2Na;
---described lipid comprises: thanomin, tween 80, Oils,glyceridic,cod-liver, cholesterol, linolic acid;
---described nucleic acid class comprises: thymidine, xanthoglobulin;
---described hormone and somatomedin class comprise: progesterone, hydrocortisone, Recombulin, putrescine;
---described antioxidant comprises: Sodium Selenite, tocopherol, taurine;
---described anti-shearing protective material is blocked polyethers F68;
---described pH indicator is phenol red;
---described pH buffer reagent comprises sodium bicarbonate, Hepes;
---described other inorganic salts comprise: calcium chloride, magnesium chloride, sal epsom, Repone K, SODIUM PHOSPHATE, MONOBASIC, sodium-chlor, Sodium phosphate dibasic, zinc sulfate, copper sulfate.
2, the animal origin-free low-protein culture medium that is suitable for the zooblast products production according to claim 1 is characterized in that, the content of various compositions is (mg/litre):
Glucose 2000～7000
Soybean hydrolyzate 500～1000
Sodium.alpha.-ketopropionate 55～220
Glutamine 200～900
L-Ala 4～15
Arginine 100～300
L-asparagine 6～15
Aspartic acid 6～15
Halfcystine 15～50
Gelucystine 30～65
L-glutamic acid 5～20
Glycine 15～40
Histidine 15～40
Isoleucine 50～100
Leucine 50～150
Methionin 80～185
Methionine(Met) 15～35
Phenylalanine-3,4-quinone 0～70
Proline(Pro) 10～34.5
Serine 20～52.5
Threonine 50～107
Tryptophane 9～18
Tyrosinase 15 5～120
Xie Ansuan 50～120
Vitamin H 0.0035～0.0105
Vitamin B12 0.50～2.50
Choline chloride 60 8.50～30
Folic acid 2.50～8
Inositol 12～30
Niacinamide 2～7
Calcium pantothenate 2～8
Vitamin B6 2～8
Riboflavin 0.2～0.7
VITMAIN B1 2～8
Thioctic Acid 0.08～0.50
Ferrous sulfate 0.417～5
Iron nitrate 0.05～0.8
EDTA·2Na 2～10
Thanomin 3～10
Tween 80 0.24～0.5
Oils,glyceridic,cod-liver 0.1～0.2
Cholesterol 0.045～0.1
Linolic acid 0.042～0.084
Thymidine 0.4～2.5
Xanthoglobulin 2～20
Progesterone 0.003～0.012
Hydrocortisone 0.036～0.072
Recombulin 3～8
Putrescine 0.08～20
Sodium Selenite 0.02～0.08
Tocopherol 1～4
Taurine 20～50
Blocked polyethers F68 500～2000
Phenol red 8.1
Sodium bicarbonate 2450
Hepes 2000～5000
Calcium chloride 110～150
Magnesium chloride 40～75
Sal epsom 45～65
Repone K 310～400
SODIUM PHOSPHATE, MONOBASIC 60～120
Sodium-chlor 6995.5
Sodium phosphate dibasic 55～120
Zinc sulfate 0.432～1.25
Copper sulfate 0.0013～0.0065.
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