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CN101603026A - Animal-source-free low-protein medium suitable for production of animal cell products - Google Patents

Animal-source-free low-protein medium suitable for production of animal cell products Download PDF

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CN101603026A
CN101603026A CNA2009100535068A CN200910053506A CN101603026A CN 101603026 A CN101603026 A CN 101603026A CN A2009100535068 A CNA2009100535068 A CN A2009100535068A CN 200910053506 A CN200910053506 A CN 200910053506A CN 101603026 A CN101603026 A CN 101603026A
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CN101603026B (en
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谭文松
赵亮
周燕
陈飞
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East China University of Science and Technology
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East China University of Science and Technology
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Abstract

本发明适于动物细胞产品生产的无动物来源低蛋白培养基，包括24种基础代谢营养物、11种维生素、3种转铁蛋白替代物类化合物、5种脂类化合物、2种核酸类化合物、4种激素及生长因子、3种抗氧化剂、1种抗剪切保护剂、1种pH指示剂、2种pH缓冲剂、9种其他无机盐，采用大豆水解物替代动物来源成分，采用硫酸亚铁、硝酸铁和EDTA·2Na的组合物替代转铁蛋白；将上述组分溶解于三次蒸馏水中，可制得所述的培养基；其积极效果是：不含动物来源成分，总蛋白含量低于10毫克/升，有利于产物的分离纯化，适于重组蛋白类药物生产，支持动物细胞的正常生长和长期传代培养，无需适应即可使用，配制方便，适于动物细胞产品的大规模生产。

The animal-source-free low-protein medium suitable for the production of animal cell products of the present invention includes 24 kinds of basal metabolic nutrients, 11 kinds of vitamins, 3 kinds of transferrin substitute compounds, 5 kinds of lipid compounds, and 2 kinds of nucleic acid compounds , 4 kinds of hormones and growth factors, 3 kinds of antioxidants, 1 kind of anti-shear protection agent, 1 kind of pH indicator, 2 kinds of pH buffering agents, 9 kinds of other inorganic salts, using soybean hydrolyzate instead of animal-derived ingredients, using sulfuric acid A composition of ferrous iron, ferric nitrate and EDTA·2Na replaces transferrin; the above-mentioned components are dissolved in triple distilled water to obtain the medium; its positive effect is: no animal-derived components, total protein content Less than 10 mg/L, which is conducive to the separation and purification of products, suitable for the production of recombinant protein drugs, supports the normal growth and long-term subculture of animal cells, can be used without adaptation, easy to prepare, and suitable for large-scale animal cell products Production.

Description

Be suitable for the animal origin-free low-protein culture medium of zooblast products production
[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、一种适于动物细胞产品生产的无动物来源低蛋白培养基，其特征在于，采用大豆水解物替代动物来源成分，采用硫酸亚铁、硝酸铁和EDTA·2Na的组合物替代转铁蛋白，其成分包括基础代谢营养物类、维生素类、转铁蛋白替代物、脂类、核酸类、激素及生长因子类、抗氧化剂类、抗剪切保护剂、pH指示剂、pH缓冲剂和其他无机盐类；1. An animal-free low-protein medium suitable for the production of animal cell products, characterized in that soybean hydrolyzate is used to replace animal-derived components, and a composition of ferrous sulfate, ferric nitrate and EDTA 2Na is used to replace transferrin , whose ingredients include basic metabolic nutrients, vitamins, transferrin substitutes, lipids, nucleic acids, hormones and growth factors, antioxidants, anti-shear protection agents, pH indicators, pH buffers and others Inorganic salts; ——所述的基础代谢营养物类包括：葡萄糖、大豆水解物、丙酮酸钠、谷氨酰胺、丙氨酸、精氨酸、天冬酰胺、天冬氨酸、半胱氨酸、胱氨酸、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸；——The basal metabolic nutrients include: glucose, soybean hydrolyzate, sodium pyruvate, glutamine, alanine, arginine, asparagine, aspartic acid, cysteine, cystine Acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine amino acid, valine; ——所述的维生素类包括：生物素、维生素、氯化胆碱、叶酸、肌醇、烟酰胺、泛酸钙、维生素B6、核黄素、维生素B1、硫辛酸；——The vitamins include: biotin, vitamins, choline chloride, folic acid, inositol, nicotinamide, calcium pantothenate, vitamin B6, riboflavin, vitamin B1, lipoic acid; ——所述的转铁蛋白替代物类包括：硫酸亚铁、硝酸铁、EDTA·2Na；——The transferrin substitutes include: ferrous sulfate, ferric nitrate, EDTA·2Na; ——所述的脂类包括：乙醇胺、吐温80、鱼肝油、胆固醇、亚油酸；——The lipids include: ethanolamine, Tween 80, cod liver oil, cholesterol, linoleic acid; ——所述的核酸类包括：胸苷、次黄嘌呤；——The nucleic acids include: thymidine, hypoxanthine; ——所述的激素及生长因子类包括：孕酮、氢化可的松、重组胰岛素、腐胺；——The hormones and growth factors include: progesterone, hydrocortisone, recombinant insulin, putrescine; ——所述的抗氧化剂类包括：亚硒酸钠、生育酚、牛磺酸；- the antioxidants include: sodium selenite, tocopherol, taurine; ——所述的抗剪切保护剂为嵌段式聚醚F68；——The anti-shear protection agent is block polyether F68; ——所述的pH指示剂为酚红；- the pH indicator is phenol red; ——所述的pH缓冲剂包括碳酸氢钠、Hepes；- the pH buffer comprises sodium bicarbonate, Hepes; ——所述的其他无机盐类包括：氯化钙、氯化镁、硫酸镁、氯化钾、磷酸二氢钠、氯化钠、磷酸氢二钠、硫酸锌、硫酸铜。——The other inorganic salts mentioned include: calcium chloride, magnesium chloride, magnesium sulfate, potassium chloride, sodium dihydrogen phosphate, sodium chloride, disodium hydrogen phosphate, zinc sulfate, copper sulfate. 2、根据权利要求1所述的适于动物细胞产品生产的无动物来源低蛋白培养基，其特征在于，各种成分的含量为(毫克/升)：2. The animal-free low-protein medium suitable for the production of animal cell products according to claim 1, characterized in that the content of various components is (mg/L): 葡萄糖       2000～7000Glucose 2000～7000 大豆水解物   500～1000Soybean hydrolyzate 500～1000 丙酮酸钠     55～220Sodium pyruvate 55～220 谷氨酰胺     200～900Glutamine 200～900 丙氨酸     4～15Alanine 4～15 精氨酸     100～300Arginine 100～300 天冬酰胺   6～15Asparagine 6～15 天冬氨酸   6～15Aspartic acid 6～15 半胱氨酸   15～50Cysteine 15～50 胱氨酸     30～65Cystine 30～65 谷氨酸     5～20Glutamic acid 5～20 甘氨酸     15～40Glycine 15～40 组氨酸     15～40Histidine 15～40 异亮氨酸   50～100Isoleucine 50～100 亮氨酸     50～150Leucine 50～150 赖氨酸     80～185Lysine 80～185 甲硫氨酸   15～35Methionine 15～35 苯丙氨酸   30～70Phenylalanine 30～70 脯氨酸     10～34.5Proline 10～34.5 丝氨酸     20～52.5Serine 20～52.5 苏氨酸     50～107Threonine 50～107 色氨酸     9～18Tryptophan 9～18 酪氨酸     55～120Tyrosine 55～120 缬氨酸     50～120Valine 50～120 生物素     0.0035～0.0105Biotin 0.0035～0.0105 维生素B12  0.50～2.50Vitamin B12 0.50～2.50 氯化胆碱   8.50～30Choline chloride 8.50～30 叶酸       2.50～8Folic acid 2.50～8 肌醇       12～30Inositol 12～30 烟酰胺     2～7Niacinamide 2～7 泛酸钙     2～8Calcium pantothenate 2～8 维生素B6   2～8Vitamin B6 2～8 核黄素     0.2～0.7Riboflavin 0.2～0.7 维生素B1   2～8Vitamin B1 2～8 硫辛酸     0.08～0.50Lipoic acid 0.08～0.50 硫酸亚铁         0.417～5Ferrous sulfate 0.417～5 硝酸铁           0.05～0.8Ferric nitrate 0.05～0.8 EDTA·2Na        2～10EDTA·2Na 2～10 乙醇胺           3～10Ethanolamine 3～10 吐温80           0.24～0.5Tween 80 0.24～0.5 鱼肝油           0.1～0.2Cod liver oil 0.1～0.2 胆固醇           0.045～0.1Cholesterol 0.045～0.1 亚油酸           0.042～0.084Linoleic acid 0.042～0.084 胸苷             0.4～2.5Thymidine 0.4～2.5 次黄嘌呤         2～20Hypoxanthine 2～20 孕酮             0.003～0.012Progesterone 0.003～0.012 氢化可的松       0.036～0.072Hydrocortisone 0.036～0.072 重组胰岛素       3～8Recombinant insulin 3～8 腐胺             0.08～20Putrescine 0.08～20 亚硒酸钠         0.02～0.08Sodium selenite 0.02～0.08 生育酚           1～4Tocopherol 1～4 牛磺酸           20～50Taurine 20～50 嵌段式聚醚F68    500～2000Block polyether F68 500～2000 酚红             8.1Phenol red 8.1 碳酸氢钠         2450Sodium bicarbonate 2450 Hepes            2000～5000Hepes 2000～5000 氯化钙           110～150Calcium chloride 110～150 氯化镁           40～75Magnesium chloride 40～75 硫酸镁           45～65Magnesium sulfate 45～65 氯化钾           310～400Potassium chloride 310～400 磷酸二氢钠       60～120Sodium dihydrogen phosphate 60～120 氯化钠           6995.5Sodium chloride 6995.5 磷酸氢二钠       55～120Disodium hydrogen phosphate 55～120 硫酸锌           0.432～1.25Zinc sulfate 0.432～1.25 硫酸铜           0.0013～0.0065。Copper sulfate 0.0013～0.0065.
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