CN101603026A

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

Info

Publication number: CN101603026A
Application number: CNA2009100535068A
Authority: CN
Inventors: 谭文松; 赵亮; 周燕; 陈飞
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2009-06-19
Filing date: 2009-06-19
Publication date: 2009-12-16
Anticipated expiration: 2029-06-19
Also published as: CN101603026B

Abstract

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

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;

——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;

——The vitamins include: biotin, vitamins, choline chloride, folic acid, inositol, nicotinamide, calcium pantothenate, vitamin B6, riboflavin, vitamin B1, lipoic acid;

——The transferrin substitutes include: ferrous sulfate, ferric nitrate, EDTA·2Na;

——The lipids include: ethanolamine, Tween 80, cod liver oil, cholesterol, linoleic acid;

——The nucleic acids include: thymidine, hypoxanthine;

——The hormones and growth factors include: progesterone, hydrocortisone, recombinant insulin, putrescine;

- the antioxidants include: sodium selenite, tocopherol, taurine;

——The anti-shear protection agent is block polyether F68;

- the pH indicator is phenol red;

- the pH buffer comprises sodium bicarbonate, Hepes;

——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. 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):

Glucose 2000～7000

Soybean hydrolyzate 500～1000

Sodium pyruvate 55～220

Glutamine 200～900

Alanine 4～15

Arginine 100～300

Asparagine 6～15

Aspartic acid 6～15

Cysteine 15～50

Cystine 30～65

Glutamic acid 5～20

Glycine 15～40

Histidine 15～40

Isoleucine 50～100

Leucine 50～150

Lysine 80～185

Methionine 15～35

Phenylalanine 30～70

Proline 10～34.5

Serine 20～52.5

Threonine 50～107

Tryptophan 9～18

Tyrosine 55～120

Valine 50～120

Biotin 0.0035～0.0105

Vitamin B12 0.50～2.50

Choline chloride 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

Vitamin B1 2～8

Lipoic acid 0.08～0.50

Ferrous sulfate 0.417～5

Ferric nitrate 0.05～0.8

EDTA·2Na 2～10

Ethanolamine 3～10

Tween 80 0.24～0.5

Cod liver oil 0.1～0.2

Cholesterol 0.045～0.1

Linoleic acid 0.042～0.084

Thymidine 0.4～2.5

Hypoxanthine 2～20

Progesterone 0.003～0.012

Hydrocortisone 0.036～0.072

Recombinant insulin 3～8

Putrescine 0.08～20

Sodium selenite 0.02～0.08

Tocopherol 1～4

Taurine 20～50

Block polyether F68 500～2000

Phenol red 8.1

Sodium bicarbonate 2450

Hepes 2000～5000

Calcium chloride 110～150

Magnesium chloride 40～75

Magnesium sulfate 45～65

Potassium chloride 310～400

Sodium dihydrogen phosphate 60～120

Sodium chloride 6995.5

Disodium hydrogen phosphate 55～120

Zinc sulfate 0.432～1.25

Copper sulfate 0.0013～0.0065.