CN102115728B

CN102115728B - Serum-free animal cell culture medium dry powder, liquid culture medium and preparation method thereof

Info

Publication number: CN102115728B
Application number: CN200910265976A
Authority: CN
Inventors: 陈文庆; 王建超; 罗海春
Original assignee: Beijing Skywing Technology Co Ltd
Current assignee: Merck Millipore Beijing Skywing Co ltd
Priority date: 2009-12-31
Filing date: 2009-12-31
Publication date: 2012-09-26
Anticipated expiration: 2029-12-31
Also published as: CN102115728A

Abstract

The invention provides a serum-free animal cell culture medium dry powder and a preparation method thereof; the serum-free animal cell culture medium dry powder comprises basic culture medium dry powder and further comprises 100-200g/100L of soy protein, 100-200g/100L of pea protein, 100-200g/100L of broad bean protein, 0-100g/100L of potato protein, 0-200g/100L of wheat gluten protein and 50-100g/100L of rice protein by taking the volume of solvent of the culture medium as reference. The invention also provides a liquid serum-free animal cell culture medium containing the serum-free animal cell culture medium dry powder and a preparation method thereof. The cells cultured by the culture medium containing the vegetable protein are high in culture density, beneficial to separating down-stream products of the cells, low in cost, small in batch difference, good in safety and suitable for producing virus host, expression vector and the like of biological products such as vaccine and the like.

Description

Serum-free animal cell culture medium dry powder, liquid nutrient medium and preparation method thereof

Technical field

The present invention relates to culture medium dry powder, liquid nutrient medium and their preparation method, relate in particular to serum-free animal cell culture medium dry powder, liquid serum-free animal cell culture medium and their preparation method.

Background technology

Prior art is through increasing the component of soya hydrolysate as serum-free animal cell culture medium; Improve cultivated host cell or reconstitution cell cultivation stability with culture density, increase the output of being cultivated host cell or reconstitution cell, and the output that increases the virus that colonizes in the host cell etc. perhaps utilize reconstitution cell as expression vector after the output of the target recombinant protein product that produces.And think the hydrolysate of other plant, for example the wheat hydrolysate can not obtain the beneficial effect suitable with soya hydrolysate.

A kind of no albumen serum free medium that is used to cultivate mammalian cell is for example disclosed among the CN 1318577C (patent No. 00816020.1); Said substratum comprises soya hydrolysate, wherein the molecular weight of at least 40% said soya hydrolysate≤500 dalton.This substratum can play stability, whole cell density and reconstitution cell target protein productive rate are cultivated in raising by cultivation of recombinant cells effect.WO02/29084 also mentions the hydrolysate that can in the substratum of culture of animal cells, add Sunlover 10 in specification sheets embodiment.

But except containing Sunlover 10, also contain many other compositions in the soya hydrolysate; Such as; VT 18, soybean polysaccharide, Soyasaponin etc.; Therefore when in serum-free animal cell culture medium, adding soya hydrolysate, very likely add above-mentioned other compositions and promptly add impurity component, thereby be unfavorable for the propagation and the growth of cell.In addition; Because Sunlover 10 is hydrolyzed the degree difference under different hydrolysising conditions, thereby the soybean protein hydrolysate composition that obtains is very difficult unified, can cause medium component uncertain; Thereby make by batch otherness of culturing cell greatly, and make by the derived product separation difficulty of culturing cell.

To sum up; Though the serum-free animal cell culture medium of soya hydrolysate has appearred adding in prior art, at present this type Zooblast culture medium have still that cell culture density is low, isolated cell derived product cost high, the defective of, poor stability big by batch otherness of culturing cell.

Summary of the invention

The objective of the invention is to overcome that existing animal cell culture serum-free animal cell culture medium exists that cell culture density is low, isolated cell derived product cost high, the shortcoming of, safe differential big by batch otherness of culturing cell; A kind of new serum-free animal cell culture medium dry powder is provided, by serum-free animal cell culture medium dry powder process liquid nutrient medium institute cultured cells culture density high, be beneficial to the isolated cell derived product, cost is low, little by batch otherness of culturing cell, security good.

Second purpose of the present invention provides the preparation method of above-mentioned serum-free animal cell culture medium dry powder.

The 3rd purpose of the present invention provides the liquid serum-free animal cell culture medium that comprises above-mentioned serum-free animal cell culture medium dry powder.

The 4th purpose of the present invention provides the preparation method of the liquid serum-free animal cell culture medium that comprises above-mentioned serum-free animal cell culture medium dry powder.

Think in the prior art that other albumen effects that in serum-free animal cell culture medium, add except that soy bean protein hydrolysate are bad; For example; Clearly put down in writing at the specification sheets 3-4 of CN 1318577C page or leaf; " opposite with known other hydrolysate of prior art (for example wheat hydrolysate, paddy rice hydrolysate or yeast), have been found that only soya hydrolysate mediation according to characteristic of the present invention, and cause for example significantly improving the yield of target recombinant protein." in the embodiment 7 of this document, disclose the wheat hydrolysate not as soybean protein hydrolysate effective.

Yet contriver of the present invention is surprised to find that; Volume with the substratum solvent in serum-free minimum medium dry powder is a benchmark; The culture medium dry powder that the Wheat bran albumen of the broad bean albumen of the Sunlover 10 of adding 100-200g/100L, the Semen Pisi sativi protein of 100-200g/100L, 100-200g/100L, the Rhizoma Solani tuber osi protein of 0-100g/100L, 0-200g/100L and the rice protein of 50-100g/100L obtain; Process liquid serum free medium culture of animal cells; Particularly when Chinese hamster ovary cell (Chinese hamster ovary celI) and hamster,syrian kidney cell (BHK21 cell), the cell culture density that can reach adds the cell culture density that the liquid serum free medium of soya hydrolysate or soybean protein hydrolysate can reach far above prior art; Because the composition that adds is confirmed, helps the isolated cell derived product on the one hand, cost is reduced, make on the other hand by batch otherness of culturing cell to reduce greatly, security is better.

The invention provides a kind of serum-free animal cell culture medium dry powder; Said culture medium dry powder is dispersed in the substratum solvent and forms liquid nutrient medium; Said culture medium dry powder comprises minimum medium dry powder; Wherein, Volume with said substratum solvent is a benchmark, and said culture medium dry powder also comprises the Sunlover 10 of 100-200g/100L, the Semen Pisi sativi protein of 100-200g/100L, the broad bean albumen of 100-200g/100L, the Rhizoma Solani tuber osi protein of 0-100g/100L, the Wheat bran albumen of 0-200g/100L and the rice protein of 50-100g/100L.

The present invention also provides the preparation method of above-mentioned serum-free animal cell culture medium dry powder, and this method comprises that the component with serum-free animal cell culture medium dry powder of the present invention is dispersed in the dispersion agent, removes the dispersion agent in the gained mixture.

The present invention also provides a kind of liquid serum-free animal cell culture medium; Said liquid serum-free animal cell culture medium comprises the substratum solvent and is dispersed in the serum-free animal cell culture medium dry powder in this substratum solvent; Wherein, said serum-free animal cell culture medium dry powder is one or more in the serum-free animal cell culture medium dry powder of the present invention.

The present invention also provides the preparation method of aforesaid liquid serum-free animal cell culture medium; This method comprises serum-free animal cell culture medium dry powder is dispersed in the substratum solvent; Filtration sterilization; Wherein, said serum-free animal cell culture medium dry powder is selected from one or more in the serum-free animal cell culture medium dry powder of the present invention.

Compare with the serum-free animal cell culture medium dry powder or the liquid serum free medium of existing interpolation soya hydrolysate or soybean protein hydrolysate; Comprise also in serum-free animal cell culture medium dry powder of the present invention or the liquid serum free medium that the volume with the substratum solvent is a benchmark, the broad bean albumen of the Sunlover 10 of 100-200g/100L, the Semen Pisi sativi protein of 100-200g/100L, 100-200g/100L, the Rhizoma Solani tuber osi protein of 0-100g/100L, the Wheat bran albumen of 0-200g/100L and the rice protein of 50-100g/100L.When serum-free animal cell culture medium dry powder of the present invention or liquid serum free medium were used for culture of animal cells, the cell culture density that can reach improved greatly; Because the composition that adds is confirmed, helps the isolated cell derived product on the one hand, cost is reduced, make on the other hand by batch otherness of culturing cell to reduce greatly, security is better.

Description of drawings

Figure 1A cultivates the photo (amplifying 420 times) of 72 hours Chinese hamster ovary celI for the serum-free animal cell culture medium dry powder that uses the embodiment of the invention 1;

Figure 1B cultivates the photo (amplifying 300 times) of 72 hours BHK21 cell for the serum-free animal cell culture medium dry powder that uses the embodiment of the invention 5;

Fig. 2 A cultivates the photo (amplifying 300 times) of 72 hours Chinese hamster ovary celI for the serum-free animal cell culture medium (comprising soya hydrolysate) that uses Comparative Examples 1 (CN 1318577C);

Fig. 2 B cultivates the photo (amplifying 300 times) of 72 hours BHK21 cell for the serum-free animal cell culture medium (comprising soya hydrolysate) that uses Comparative Examples 1 (CN 1318577C);

Fig. 3 A cultivates the photo (amplifying 300 times) of 72 hours Chinese hamster ovary celI for the serum-free animal cell culture medium (comprising soya hydrolysate) that uses Comparative Examples 2 (WO 02/29084);

Fig. 3 B cultivates the photo (amplifying 300 times) of 72 hours BHK21 cell for the serum-free animal cell culture medium (comprising soya hydrolysate) that uses Comparative Examples 2 (WO 02/29084);

Fig. 4 A cultivates the photo (amplifying 420 times) of 72 hours Chinese hamster ovary celI for the liquid serum-free animal cell culture medium that uses the embodiment of the invention 2;

Fig. 4 B cultivates the photo (amplifying 300 times) of 72 hours BHK21 cell for the liquid serum-free animal cell culture medium that uses the embodiment of the invention 6;

Fig. 5 A cultivates the photo (amplifying 420 times) of 48 hours Chinese hamster ovary celI for the cultivation liquid serum-free animal cell culture medium that uses embodiment 3;

Fig. 5 B cultivates the photo (amplifying 300 times) of 48 hours BHK21 cell for the cultivation liquid serum-free animal cell culture medium that uses embodiment 7;

Fig. 6 A cultivates the photo (amplifying 420 times) of 48 hours Chinese hamster ovary celI for the cultivation liquid serum-free animal cell culture medium that uses embodiment 4;

Fig. 6 B cultivates the photo (amplifying 200 times) of 48 hours BHK21 cell for the cultivation liquid serum-free animal cell culture medium that uses embodiment 8;

Fig. 7 A is the Chinese hamster ovary celI density comparison diagram that Comparative Examples 1-2 and embodiment 1-4 change with incubation time;

Fig. 7 B is the BHK21 cell density comparison diagram that Comparative Examples 1-2 and embodiment 5-8 change with incubation time.

Embodiment

Serum-free animal cell culture medium dry powder provided by the invention; Said culture medium dry powder is dispersed in the substratum solvent and forms liquid nutrient medium; Said culture medium dry powder comprises minimum medium dry powder; Wherein, Volume with said substratum solvent is a benchmark, and said culture medium dry powder also comprises the Sunlover 10 of 100-200g/100L, the Semen Pisi sativi protein of 100-200g/100L, the broad bean albumen of 100-200g/100L, the Rhizoma Solani tuber osi protein of 0-100g/100L, the Wheat bran albumen of 0-200g/100L and the rice protein of 50-100g/100L.

Serum-free animal cell culture medium dry powder of the present invention is benchmark with the volume of said substratum solvent wherein preferably, and said culture medium dry powder also comprises the Sunlover 10 of 150-180g/100L, the Semen Pisi sativi protein of 150-180g/100L, the broad bean albumen of 150-180g/100L, the Rhizoma Solani tuber osi protein of 50-80g/100L, the Wheat bran albumen of 150-180g/100L and the rice protein of 50-80g/100L.Serum-free animal cell culture medium dry powder of the present invention is benchmark with the volume of said substratum solvent wherein more preferably, and said culture medium dry powder also comprises the Sunlover 10 of 170-180g/100L, the Semen Pisi sativi protein of 170-180g/100L, the broad bean albumen of 170-180g/100L, the Rhizoma Solani tuber osi protein of 50-60g/100L, the Wheat bran albumen of 150-160g/100L, the rice protein of 50-60g/100L.

In the serum-free animal cell culture medium dry powder of the present invention; Said Sunlover 10, Semen Pisi sativi protein, broad bean albumen, Rhizoma Solani tuber osi protein, Wheat bran albumen and rice protein can be the commercial commodity that supply, and also can be the albumen of from corresponding plant seed or fruit, separating according to the normal experiment means.The detection method of proteinic separating and purifying method and qualitative, quantitative is as well known to those skilled in the art; Such as TCA-acetone method (Mechin V; Damerval C, Zivy M.Total protein extraction with TCA-acetone.Methods MolBiol 2007; 355:1-8), acid hydrolysis and enzyme hydrolysis method, the preferred enzyme hydrolysis method.

Preferred Sunlover 10 of the present invention, Semen Pisi sativi protein, broad bean albumen, Rhizoma Solani tuber osi protein, Wheat bran albumen and rice protein prepare through following method:

A) milling soya seeds, pea, broad bean, yam, Wheat bran or rice plants raw material to diameter are 0.1-3mm ³Particle;

B) be that 0.5-10h, enzymolysis pH value are that 5-9, hydrolysis temperature are that 45 ℃-60 ℃, enzyme amount are a) particle of gained of enzymolysis step under the condition of 2-5 weight % at enzymolysis time; Wherein, said enzyme is selected from one or more in trypsinase, neutral protease, Sumizyme MP, papoid and the bromeline, and the enzyme of said enzyme lives>=1 * 10 ⁵Units/gram;

C) keep 5-20min to stop the described enzymolysis of step b) at 80-100 ℃;

D) with enzymolysis product centrifugal 5-20min under 3000-8000rpm of step c) gained, collect supernatant;

E) step d) gained supernatant is used the hollow fiber filter ultrafiltration, the molecular weight that sees through of the used film of said strainer is 10000-20000 dalton;

F) collect step e) gained ultrafiltrated, vacuum lyophilization obtains corresponding vegetable-protein.

More preferably said Sunlover 10, Semen Pisi sativi protein, broad bean albumen, Rhizoma Solani tuber osi protein, Wheat bran albumen and rice protein prepare through following method:

A) milling soya seeds, pea, broad bean, yam, Wheat bran or rice plants raw material to diameter are 0.1-1mm ³Particle;

B) be that 2-10h, enzymolysis pH value are that 5-8, hydrolysis temperature are that 45 ℃-60 ℃, enzyme amount are a) particle of gained of enzymolysis step under the condition of 2-5 weight % at enzymolysis time; Wherein, said enzyme is selected from one or more in trypsinase, neutral protease, Sumizyme MP, papoid and the bromeline, and the enzyme of said enzyme lives>=1 * 10 ⁵Units/gram;

C) keep 5-10min to stop the described enzymolysis of step b) at 80-90 ℃;

D) with enzymolysis product centrifugal 15-20min under 3000-5000rpm of step c) gained, collect supernatant;

More preferably, carry out different pretreatment according to different plant materials, for example, can first degreasing pre-treatment for bean material (soybean, pea and bean).Different plant materials also can adopt different enzymatic hydrolysis conditions, for example, is 52.9 ℃, Sumizyme MP (10 in temperature ⁵Units/gram) enzyme concentration is 5.2%, pH is 8.7, concentration of substrate is 8.06 weight % and extraction time to be preparation Wheat bran albumen under 2 hours the condition; Be 50 ℃, neutral protease (10 perhaps in temperature ⁵Units/gram) enzyme concentration is 4%, pH is 6.8, concentration of substrate is 6.5 weight % and extraction time to be preparation Wheat bran albumen under 2.5 hours the condition.Rhizoma Solani tuber osi protein can make through following method: clean peeling raw potatoes and equal-volume 1% sodium sulfite solution homogenate; Solid-liquid separation (centrifugal, leave standstill or squeeze the juice); Collecting the gained potato juice is to leave standstill 5h 3.0 times at pH, centrifugal collecting precipitation, and spraying drying gets powder.Collecting said powder with damping fluid, is 50 ℃, trypsinase enzyme concentration (10 in temperature ⁵Units/gram) be 4%, pH is 8.0, concentration of substrate is 20 weight % (10 ⁵Units/gram) and extraction time be to prepare Rhizoma Solani tuber osi protein under 1.5 hours the condition.

Serum-free animal cell culture medium dry powder of the present invention can comprise the various minimum mediums that this area is commonly used; For example, be selected from BME cell culture medium, DMEM cell culture medium, DMEM/F12 cell culture medium, Fischer ' s cell culture medium, IMDM cell culture medium, 199 cell culture mediums, MEM cell culture medium, F10 cell culture medium, F12 cell culture medium and 1640 cell culture mediums one or more.The said minimum medium that preferably includes is 1: 1 F12 cell culture medium of weight ratio and DMEM cell culture medium.The composition of said minimum medium is known in this field, and the above-mentioned minimum medium name of enumerating is called their trade(brand)name, the commercial confession.

Serum-free animal cell culture medium dry powder more preferably of the present invention wherein, is benchmark with the volume of said substratum solvent, and said serum-free animal cell culture medium dry powder comprises the component shown in the following table 1:

Table 1

Component	g/100L	Component	g/100L
				Calcium Chloride Powder Anhydrous	10-20	The L-tryptophane	0.5-2
Salzburg vitriol	0-0.001	L-tyrosine	2-10
				Nine nitric hydrate iron	0-0.01	The L-Xie Ansuan	3-10
Presfersul	0.1-0.5	D-glucose	500-700
				Repone K	10-50	HEPES	300-400
Magnesium dichloride hexahydrate	10-20	Linolic acid	0.01-0.05
				Anhydrous magnesium sulfate	2-8	Thioctic Acid	0.05-0.1
Sodium-chlor	600-700	1,4-tetramethylenediamine dihydrochloride	0.02-1
				AMSP	2-8	Sodium.alpha.-ketopropionate	10-20
Sodium phosphate, dibasic	6-10	Vitamin H	0.001-0.005
				Zinc vitriol	0.01-0.5	The D-VA	0.1-0.5
Anhydrous potassium dihydrogenphosphate	2-10	Choline chloride 60	5-10
				The L-arginine monohydrochloride	20-100	Folic acid	0.1-0.5
The L-cystine hydrochloride	3-10	The i-inositol	2-10
				L-glutaminate	50-200	Vitamin PP	0.1-0.5
Glycocoll	2-8	Pyridoxal hydrochloride	0.1-0.5
				The L-L-Histidine hydrochloride	1-5	Pyridoxine hydrochloride	0.01-0.05
The L-Isoleucine	4-10	Vitamin G	0.01-0.05
				The L-leucine	4-10	Thiamine hydrochloride	0.1-0.5
L lysine HCL	5-12	Thymidine	0.01-0.05
				The L-methionine(Met)	0.5-5	Cobalamin	0.1-1
The L-phenylalanine(Phe)	2-6	The recombinant human insulin	0.2-1
				The L-Serine	1.5-5	Selenous acid	0.0001-0.001
The L-Threonine	3-8	Thanomin	0-0.0005
				The L-L-Ala	0.2-1	Sunlover 10	100-200
The L-asparagine	6-15	Semen Pisi sativi protein	100-200
				The L-aspartic acid	0.5-2	Broad bean albumen	100-200
The L-cysteine hydrochloride	10-20	Rhizoma Solani tuber osi protein	0-100
				L-L-glutamic acid	0-2	Wheat bran albumen	0-200
The L-proline(Pro)	8-20	Rice protein	50-100

More preferably described serum-free animal cell culture medium dry powder is benchmark with the volume of said substratum solvent, and said serum-free animal cell culture medium dry powder can also comprise xanthoglobulin and/or 0.1-2g/100L phenol red of 0.1-1g/100L.Wherein, xanthoglobulin can be used as the source of purine; Phenol red as the adding of pH pH indicator, can be through estimating the growing state that grasp cell at any time.

Further preferred described serum-free animal cell culture medium dry powder wherein, is benchmark with the volume of said substratum solvent, and said serum-free animal cell culture medium dry powder comprises the component shown in the following table 2:

Table 2

Component	g/100L	Component	g/100L
				Calcium Chloride Powder Anhydrous	10-15	The L-tryptophane	1-1.5
Salzburg vitriol	0-0.0005	L-tyrosine	3-8
				Nine nitric hydrate iron	0.002-0.01	The L-Xie Ansuan	4-9
Presfersul	0.1-0.4	D-glucose	550-680
				Repone K	20-40	HEPES	350-380
Magnesium dichloride hexahydrate	1-20	Linolic acid	0.01-0.04
				Anhydrous magnesium sulfate	3-7	Thioctic Acid	0.05-0.08
Sodium-chlor	650-700	1,4-tetramethylenediamine dihydrochloride	0.05-0.5
				AMSP	3-8	Sodium.alpha.-ketopropionate	12-18
Sodium phosphate, dibasic	6-9	Vitamin H	0.001-0.004
				Zinc vitriol	0.01-0.5	The D-VA	0.1-0.4
Anhydrous potassium dihydrogenphosphate	3-8	Choline chloride 60	6-9
				The L-arginine monohydrochloride	20-100	Folic acid	0.1-0.4
The L-cystine hydrochloride	3-7	The i-inositol	3-9
				L-glutaminate	50-200	Vitamin PP	0.1-0.4
Glycocoll	3-7	Pyridoxal hydrochloride	0.1-0.4
				The L-L-Histidine hydrochloride	3-5	Pyridoxine hydrochloride	0.01-0.04
The L-Isoleucine	4-8	Vitamin G	0.01-0.04
				The L-leucine	4-8	Thiamine hydrochloride	0.1-0.4
L lysine HCL	8-12	Thymidine	0.01-0.04
				The L-methionine(Met)	1-4	Cobalamin	0.3-0.9
The L-phenylalanine(Phe)	3-5	The recombinant human insulin	0.3-0.9
				The L-Serine	2-4	Selenous acid	0.0002-0.0008
The L-Threonine	4-7	Thanomin	0.0001-0.0004
				The L-L-Ala	0.5-0.8	Sunlover 10	150-180
The L-asparagine	8-12	Semen Pisi sativi protein	150-180
				The L-aspartic acid	1-2	Broad bean albumen	150-180
The L-cysteine hydrochloride	10-15	Rhizoma Solani tuber osi protein	50-80
				L-L-glutamic acid	0.5-1.5	Wheat bran albumen	150-180
The L-proline(Pro)	10-15	Rice protein	50-80

When said serum-free animal cell culture medium dry powder is when cultivating the culture medium dry powder of Chinese hamster ovary cell; Be that serum-free animal cell culture medium dry powder of the present invention is when being used to cultivate Chinese hamster ovary celI; In the preferred described serum-free animal cell culture medium dry powder; Volume with said substratum solvent is a benchmark, comprises the component shown in the following table 3:

Table 3

Component	g/100L	Component	g/100L
				Calcium Chloride Powder Anhydrous	10-20	The L-tryptophane	0.5-2
Salzburg vitriol	0-0.001	L-tyrosine	2-10
				Nine nitric hydrate iron	0-0.01	The L-Xie Ansuan	3-10
Presfersul	0.1-0.5	D-glucose	500-700
				Repone K	10-50	HEPES	300-400
Magnesium dichloride hexahydrate	10-20	Linolic acid	0.01-0.05
				Anhydrous magnesium sulfate	2-8	Thioctic Acid	0.05-0.1
Sodium-chlor	600-700	1,4-tetramethylenediamine dihydrochloride	0.02-1
				AMSP	2-8	Sodium.alpha.-ketopropionate	10-20
Sodium phosphate, dibasic	6-10	Vitamin H	0.001-0.005
				Zinc vitriol	0.1-0.5	The D-VA	0.1-0.5
Anhydrous potassium dihydrogenphosphate	2-10	Choline chloride 60	5-10
				The L-arginine monohydrochloride	50-100	Folic acid	0.1-0.5
The L-cystine hydrochloride	3-10	The i-inositol	2-10
				L-glutaminate	100-200	Vitamin PP	0.1-0.5
Glycocoll	2-8	Pyridoxal hydrochloride	0.1-0.5
				The L-L-Histidine hydrochloride	1-5	Pyridoxine hydrochloride	0.01-0.05
The L-Isoleucine	4-10	Vitamin G	0.01-0.05
				The L-leucine	4-10	Thiamine hydrochloride	0.1-0.5
L lysine HCL	5-12	Thymidine	0.01-0.05
				The L-methionine(Met)	0.5-5	Cobalamin	0.1-1
The L-phenylalanine(Phe)	2-6	The recombinant human insulin	0.2-1
				The L-Serine	1.5-5	Selenous acid	0.0001-0.001
The L-Threonine	3-8	Thanomin	0-0.0005
				The L-L-Ala	0.2-1	Sunlover 10	100-200
The L-asparagine	6-15	Semen Pisi sativi protein	100-180
				The L-aspartic acid	0.5-2	Broad bean albumen	100-200
The L-cysteine hydrochloride	10-20	Rhizoma Solani tuber osi protein	50-100
				L-L-glutamic acid	0-2	Wheat bran albumen	150-180
The L-proline(Pro)	8-20	Rice protein	50-100

When said serum-free animal cell culture medium dry powder is when cultivating the culture medium dry powder of BHK21 cell; Be that serum-free animal cell culture medium dry powder of the present invention is when being used to cultivate the BHK21 cell; In the preferred described serum-free animal cell culture medium dry powder; Volume with said substratum solvent is a benchmark, comprises the component shown in the following table 4:

Table 4

Component	g/100L	Component	g/100L
				Calcium Chloride Powder Anhydrous	10-20	The L-tryptophane	0.5-2
Salzburg vitriol	0-0.001	L-tyrosine	2-10
				Nine nitric hydrate iron	0-0.01	The L-Xie Ansuan	3-10
Presfersul	0.1-0.5	D-glucose	500-700
				Repone K	10-50	HEPES	300-400
Magnesium dichloride hexahydrate	1-10	Linolic acid	0.01-0.05
				Anhydrous magnesium sulfate	2-8	Thioctic Acid	0.05-0.1
Sodium-chlor	600-700	1,4-tetramethylenediamine dihydrochloride	0.02-1
				AMSP	2-8	Sodium.alpha.-ketopropionate	10-20
Sodium phosphate, dibasic	6-10	Vitamin H	0.001-0.005
				Zinc vitriol	0.01-0.05	The D-VA	0.1-0.5
Anhydrous potassium dihydrogenphosphate	2-10	Choline chloride 60	5-10
				The L-arginine monohydrochloride	20-80	Folic acid	0.1-0.5
The L-cystine hydrochloride	3-10	The i-inositol	2-10
				L-glutaminate	50-150	Vitamin PP	0.1-0.5
Glycocoll	2-8	Pyridoxal hydrochloride	0.1-0.5
				The L-L-Histidine hydrochloride	1-5	Pyridoxine hydrochloride	0.01-0.05
The L-Isoleucine	4-10	Vitamin G	0.01-0.05
				The L-leucine	4-10	Thiamine hydrochloride	0.1-0.5
L lysine HCL	5-12	Thymidine	0.01-0.05
				The L-methionine(Met)	0.5-5	Cobalamin	0.1-1
The L-phenylalanine(Phe)	2-6	The recombinant human insulin	0.2-1
				The L-Serine	1.5-5	Selenous acid	0.0001-0.001
The L-Threonine	3-8	Thanomin	0-0.0005
				The L-L-Ala	0.2-1	Sunlover 10	100-200
The L-asparagine	6-15	Semen Pisi sativi protein	120-200
				The L-aspartic acid	0.5-2	Broad bean albumen	120-180
The L-cysteine hydrochloride	10-20	Rhizoma Solani tuber osi protein	0-50
				L-L-glutamic acid	0-2	Wheat bran albumen	0-100
The L-proline(Pro)	8-20	Rice protein	50-100

The present invention also provides the preparation method of above-mentioned serum-free animal cell culture medium dry powder, and this method comprises that the component with serum-free animal cell culture medium dry powder of the present invention is dispersed in the dispersion agent, removes the dispersion agent in the gained mixture.The dispersion of the component of said serum-free animal cell culture medium dry powder in dispersion agent can realize through this area various dispersing method commonly used, such as stirring, sonic oscillation etc.

Prepare serum-free animal cell culture medium dry powder of the present invention and can use dry method and wet method two class methods.So-called dry method refers to the powder (such as utilizing the ball mill ball mill mixing) of blended solid component under the exsiccant condition.When using dry method, preferred at first dry respectively based on the character of each component of powder to their according to constituting cultivation.Such as can be at 100-120 ℃ with AMSP, Repone K, anhydrous magnesium sulfate, sodium-chlor, Sodium phosphate, dibasic, anhydrous potassium dihydrogenphosphate, preferred 100-105 ℃ dry 1-5 hour down; L-L-Ala, L-aspartic acid, L-L-glutamic acid, L-glutaminate, L-L-Histidine hydrochloride, L-Isoleucine, L-leucine, L lysine HCL, L-methionine(Met), L-phenylalanine(Phe), L-proline(Pro), L-Serine, L-tryptophane, L-Threonine, L-Xie Ansuan, L-tyrosine, glycocoll, L-cystine hydrochloride were descended dry 1-3 hour at 30-80 ℃ of preferred 35-50 ℃; Presfersul, Zinc vitriol, Sodium.alpha.-ketopropionate are put into vacuum drying oven to vacuumize dry more than 10 hours.So-called wet method refers to form the solid ingredient and the dispersant of serum-free animal cell culture medium dry powder of the present invention, removes the dispersion agent of gained mixture then.Can also dry method be combined with wet method, be about to a part of solid ingredient and use wet-mixed, remove dispersion agent after, mix with the solid ingredient of rest part with dry method.

Said dispersion agent can be selected from the various dispersion agents that this area is used to prepare serum-free animal cell culture medium dry powder.The effect of said dispersion agent is that the various components of serum-free animal cell culture medium dry powder are mixed equably; Generally dispersion agent there is not special requirement; Only otherwise introduce the new ion that influences the cell growth, pollute impurity (like intracellular toxin, mikrobe etc.), do not change pH value scope that this Zooblast culture medium dry powder is scattered in the suitable cell growth behind the substratum solvent, removal gets final product easily under the prerequisite of not destroying nutrient media components (such as making protein denaturation).Therefore said dispersion agent be preferably selected from zero(ppm) water, deionized water, water for injection, redistilled water, ultrapure water, in alcohol, alkaline aqueous solution and the acidic aqueous solution that can dissolve each other with any ratio with water one or more; Wherein said alkaline aqueous solution is the aqueous solution of the cationic alkali that occurs in ammoniacal liquor or the serum-free animal cell culture medium dry powder of the present invention, the aqueous acid of the acid ion that said acidic aqueous solution occurs for serum-free animal cell culture medium dry powder among the present invention.Said can be in ethanol, methyl alcohol, the propyl alcohol one or more with the alcohol that water dissolves each other with any ratio.In the aqueous solution of the aqueous solution of the aqueous solution of the preferred sodium hydroxide of said alkaline aqueous solution, the aqueous solution of Pottasium Hydroxide, calcium hydroxide, ammoniacal liquor, basic aminoacids one or more, the most preferably aqueous solution of sodium hydroxide.In the aqueous solution of the aqueous solution of said acidic aqueous solution preferably salt acid solution, the vitriolic aqueous solution, nitric acid, organic acid (carbonic acid, oxalic acid, succsinic acid, oxysuccinic acid, pyruvic acid, phosphoric acid, acidic amino acid) one or more, most preferably aqueous solution of hydrochloric acid.

The present invention does not have special requirement to the order that each component in the said serum-free animal cell culture medium dry powder joins in the dispersion agent.In addition, can various components be dispersed in in a kind of dispersion agent, also can different components be dispersed in the different dispersion agents, mix the dispersion-s that obtains then.The preferred latter for example preferably is scattered in vitamin H, thymidine, xanthoglobulin in the aqueous solution (aqueous solution of sodium hydroxide is preferably prepared with redistilled water) of the sodium hydroxide of 1-10 weight %, can suitably be heated to 60 ℃; It is in 1 the acidic aqueous solution (like the aqueous hydrochloric acid with the redistilled water preparation) that tyrosine is scattered in the pH value; Lipophilic substance (like linolic acid) is scattered in earlier in the hydrophilic small molecules organic solvent of lower boiling (like ethanol).For the component (being no more than the component of 0.01g/100L such as content) seldom of content in substratum; In the preparation; Can according to the required quantitative form of this component in the final culture medium dry powder this component be mixed with other components then earlier with the high concentration liquid dispersion-s of this component of dispersion agent preparation with this high concentration liquid dispersion-s.The temperature of used dispersion agent should not be higher than by the lowest decomposition temperature of dispersed component.

The consumption of said dispersion agent has no particular limits, as long as can make the component of culture medium dry powder and the dispersion-s that dispersion agent forms stable homogeneous; In addition, satisfying under the prerequisite of aforementioned condition, from the consideration of energy consumption, the consumption of dispersion agent is few more good more.

Can adopt this area method commonly used to remove dispersion agent, such as drying.Said drying can be heating, vacuum-drying, vacuum lyophilization, siccative (like silica gel, alumina gel, molecular sieve, gac, bone black, charcoal, atlapulgite, five phosphorus oxide, unslaked lime, calcium chloride etc.) moisture absorption etc.

Substratum solvent described in the liquid serum-free animal cell culture medium of the present invention can be selected from the various substratum solvents of this area as culturing cell; Be preferably selected from zero(ppm) water, deionized water, water for injection, redistilled water and the ultrapure water one or more, more preferably said substratum solvent is water for injection and/or ultrapure water.

The present invention also provides the preparation method of aforesaid liquid serum-free animal cell culture medium; This method comprises serum-free animal cell culture medium dry powder is dispersed in the substratum solvent; Filtration sterilization; Wherein, said serum-free animal cell culture medium dry powder is selected from one or more in the serum-free animal cell culture medium dry powder of the present invention.The component that the preparation method of aforesaid liquid serum-free animal cell culture medium also can be liquid serum-free animal cell culture medium of the present invention directly is dispersed in the substratum solvent with solid form one by one.For the component (being no more than the component of 0.01g/100L such as content) seldom of content in the said liquid serum-free animal cell culture medium; In the preparation; Can add this component according to the required quantitative form of component in the final liquid nutrient medium then earlier with the high concentration liquid dispersion-s of this component of substratum solvent preparation with this high concentration liquid dispersion-s.The temperature of used substratum solvent should not be higher than by the lowest decomposition temperature of dispersed component.

The method of said filtration sterilization is known in this field, can adopt the aperture to be not more than the bacteriological filtration film of 0.22 μ m, carries out once or filtration several times.The dispersion of said serum-free animal cell culture medium dry powder in the substratum solvent can realize through this area various dispersing method commonly used, such as stirring, sonic oscillation etc.

Serum-free animal cell culture medium dry powder of the present invention, liquid serum-free animal cell culture medium are to can cultured cells having no particular limits; Can cultivate somatocyte (like the Vero cell) or sexual cell (Chinese hamster ovary celI); Primary cell (like hamster kidney cell) or passage cell (RhMK MA-104); The cell (hybridoma) that cancer cells such as hela cell, normal cell and process genetically engineered are modified or the cell of process artificially breeding are (like the BHK21 cell; Be the hamster,syrian kidney cell, claim the cricetulus auratus kidney cell again).Serum-free animal cell culture medium dry powder of the present invention, liquid serum-free animal cell culture medium also have no particular limits training method, can be used for adherent culture, suspension culture and immobilization and cultivate.Be more suitable for being used in the suspension culture process of Chinese hamster ovary celI; Also be more suitable for being used in the BHK21 suspension culture of cells process.Chinese hamster ovary celI can be used as the expression vector of multiple antibody.The BHK21 cell can be used as the host cell of multiple virus.Can adopt the conventional condition in this area to carry out animal cell culture, for example, the inoculum size of cell can be 10 in the suspension culture process ³-10 ⁸Individual/the milliliter substratum, the inoculum size that is preferably cell is 10 ⁵-10 ⁶Individual/the milliliter substratum; The temperature of cultivating can be 30-38 ℃, and humidity can be 3-8 volume % for 90-98% and concentration of carbon dioxide, and preferably including temperature is 35-37 ℃, and humidity is that 92-96% and concentration of carbon dioxide are 4-6 volume %.Wherein, when the suspension culture Chinese hamster ovary cell, the inoculum size of cell can be 10 ³-10 ⁸Individual/the milliliter substratum; The temperature of cultivating can be 30-38 ℃, and humidity can be 3-8 volume % for 90-98% and concentration of carbon dioxide.Wherein, when suspension culture BHK21 cell, the inoculum size of cell can be 10 ³-10 ⁸Individual/the milliliter substratum; The temperature of cultivating can be 30-38 ℃, and humidity can be 3-8 volume % for 90-98% and concentration of carbon dioxide.

Unless stated otherwise, all ingredients that the present invention uses all can be purchased, and also can adopt this area method preparation commonly used.The reagent that is used for cell cultures, not only purity requirement is high, preferred analytical pure, and can not have any material that influences the cell normal physiological activity, therefore also need meet the requirement of pharmaceutical products, preferred injection stage bulk drug.

Below in conjunction with embodiment, further specify the present invention.

Embodiment 1

Present embodiment is used to explain serum-free animal cell culture medium dry powder of the present invention and preparation method thereof, and liquid serum-free animal cell culture medium of the present invention and preparation method thereof.

Table 5

Component	g/100L	Component	g/100L
				Calcium Chloride Powder Anhydrous	12	The L-tryptophane	0.5
Salzburg vitriol	0.001	L-tyrosine	5
				Nine nitric hydrate iron	0.003	The L-Xie Ansuan	3
Presfersul	0.5	D-glucose	500
				Repone K	35	HEPES	300
Magnesium dichloride hexahydrate	20	Linolic acid	0.04
				Anhydrous magnesium sulfate	5	Thioctic Acid	0.05
Sodium-chlor	680	1,4-tetramethylenediamine dihydrochloride	0.02
				AMSP	4	Sodium.alpha.-ketopropionate	10
Sodium phosphate, dibasic	7	Vitamin H	0.001
				Zinc vitriol	0.5	The D-VA	0.1
Anhydrous potassium dihydrogenphosphate	4	Choline chloride 60	10
				The L-arginine monohydrochloride	80	Folic acid	0.5
The L-cystine hydrochloride	5	The i-inositol	2
				L-glutaminate	190	Vitamin PP	0.5
Glycocoll	4	Pyridoxal hydrochloride	0.1
				The L-L-Histidine hydrochloride	5	Pyridoxine hydrochloride	0.02
The L-Isoleucine	6	Vitamin G	0.01
				The L-leucine	6	Thiamine hydrochloride	0.1
L lysine HCL	11	Thymidine	0.01
				The L-methionine(Met)	3	Cobalamin	0.1
The L-phenylalanine(Phe)	4.5	The recombinant human insulin	0.2
				The L-Serine	4	Selenous acid	0.0001
The L-Threonine	6	Thanomin	0.0004
				The L-L-Ala	0.5	Sunlover 10	100
The L-asparagine	11	Semen Pisi sativi protein	100
				The L-aspartic acid	1.5	Broad bean albumen	100
The L-cysteine hydrochloride	13	Rhizoma Solani tuber osi protein	100
				L-L-glutamic acid	1	Wheat bran albumen	150
The L-proline(Pro)	14	Rice protein	50

The g/100L of unit of solid ingredient is meant in table 5 and following each table; Volume to prepare the required substratum solvent that uses of final liquid nutrient medium is benchmark; With respect to per 100 liters of substratum solvents, the gram number of the solid matter that when the final liquid nutrient medium of preparation, need add.With the consumption of the solid ingredient shown in the table 5 according to 100 liters of final liquid nutrient mediums; After accurately weighing mixes one by one with ten thousand/balance, add 50 liters of zero(ppm) water again, with the stirring of Mei Ying Pu, the Shanghai 85-2 of instrument Manufacturing Co., Ltd type whisking appliance after 2 hours; Obtain homogeneous dispersion; Use the medium-sized freeze drier of FD-3A (Xi'an moral growth Instr Ltd.), according to this instrument specification sheets, at precooling temperature-45 ℃; Lyophilize is 50 hours under the slow interior vacuum tightness 9Pa of maintenance instrument that heats up, and obtains serum-free animal cell culture medium dry powder of the present invention.Serum-free animal cell culture medium dry powder seal at normal temperatures keep in Dark Place subsequent use.

With the above-mentioned serum-free animal cell culture medium dry powder of the present invention that makes 2565.756 grams, be dissolved in 30 ℃ of 100L ultrapure waters, gained solution uses the bacteriological filtration membrane filtration sterilization of 0.20 μ m, promptly gets the liquid serum-free animal cell culture medium, and refrigeration is subsequent use down at 2 ℃.

Embodiment 2

Table 6

Component	g/100L	Component	g/100L
				Calcium Chloride Powder Anhydrous	12	The L-tryptophane	2
Salzburg vitriol	0.001	L-tyrosine	8
				Nine nitric hydrate iron	0	The L-Xie Ansuan	10
Presfersul	0.1	D-glucose	550
				Repone K	30	HEPES	320
Magnesium dichloride hexahydrate	15	Linolic acid	0.02
				Anhydrous magnesium sulfate	6	Thioctic Acid	0.1
Sodium-chlor	640	1,4-tetramethylenediamine dihydrochloride	0.09
				AMSP	8	Sodium.alpha.-ketopropionate	14
Sodium phosphate, dibasic	7	Vitamin H	0.005
				Zinc vitriol	0.5	The D-VA	0.4
Anhydrous potassium dihydrogenphosphate	8	Choline chloride 60	5
				The L-arginine monohydrochloride	100	Folic acid	0.1
The L-cystine hydrochloride	7	The i-inositol	4
				L-glutaminate	140	Vitamin PP	0.1
Glycocoll	2	Pyridoxal hydrochloride	0.3
				The L-L-Histidine hydrochloride	1	Pyridoxine hydrochloride	0.04
The L-Isoleucine	4	Vitamin G	0.05
				The L-leucine	4	Thiamine hydrochloride	0.5
L lysine HCL	5	Thymidine	0.05
				The L-methionine(Met)	3	Cobalamin	1
The L-phenylalanine(Phe)	6	The recombinant human insulin	0.4
				The L-Serine	5	Selenous acid	0.001
The L-Threonine	7	Thanomin	0.0002
				The L-L-Ala	0.7	Sunlover 10	200
The L-asparagine	6	Semen Pisi sativi protein	120
				The L-aspartic acid	1.5	Broad bean albumen	140
The L-cysteine hydrochloride	20	Rhizoma Solani tuber osi protein	50
				L-L-glutamic acid	0.5	Wheat bran albumen	180
The L-proline(Pro)	12	Rice protein	100
				Xanthoglobulin	0.3

Solid ingredient shown in the table 6 takes by weighing with ten thousand/analytical balance according to the consumption of 100 liters of final liquid nutrient mediums.Wherein, vitamin H, xanthoglobulin, thymidine are dissolved in the 1mol/L NaOH distilled water solution of 1000ml, obtain A solution; With the L-L-Ala; The L-aspartic acid; L-L-glutamic acid; L-glutaminate; The L-L-Histidine hydrochloride; L lysine HCL; The L-arginine monohydrochloride; The L-Isoleucine; The L-leucine; The L-methionine(Met); The L-phenylalanine(Phe); The L-proline(Pro); The L-Serine; The L-tryptophane; The L-Threonine; The L-Xie Ansuan; Choline chloride 60; The D-VA; Folic acid; Vitamin PP; Pyridoxine hydrochloride; Vitamin G; Thiamine hydrochloride; Cobalamin; Pyridoxal hydrochloride; The recombinant human insulin; Selenous acid is dissolved in and obtains B solution in the 3000ml zero(ppm) water; The ethanol that linolic acid is dissolved in 10ml 99% obtains C solution; It is to obtain D solution in 1 the hydrochloric acid distilled water solution that L-tyrosine is scattered in the pH value; Merge A solution, B solution, C solution and D solution; In the gained mixing solutions, add glucose, AMSP, Repone K, anhydrous magnesium sulfate, Calcium Chloride Powder Anhydrous, sodium-chlor, ADSP, anhydrous potassium dihydrogenphosphate, glycocoll, L-cystine hydrochloride, L-cysteine hydrochloride, L-asparagine, Presfersul, Zinc vitriol, Sodium.alpha.-ketopropionate, cupric sulfate pentahydrate, nine water iron nitrates, HEPES, 1 then; 4-tetramethylenediamine dihydrochloride, i-inositol, thanomin, Sunlover 10, Semen Pisi sativi protein, broad bean albumen, Rhizoma Solani tuber osi protein, Wheat bran albumen, rice protein; Being settled to 10000ml with zero(ppm) water shakes up; Collect the gained mixed solution; Use the medium-sized freeze drier of FD-3A (Xi'an moral growth Instr Ltd.), according to this instrument specification sheets, at precooling temperature-50 ℃; Lyophilize is 40 hours under the maintenance vacuum tightness that slowly the heats up 10Pa, promptly obtains serum-free animal cell culture medium dry powder.Serum-free animal cell culture medium dry powder seal at normal temperatures keep in Dark Place subsequent use.

With the above-mentioned Zooblast culture medium dry powder that makes 2757.757 grams, be dissolved in the 80L ultrapure water, be settled to 100L with ultrapure water then.Use the bacteriological filtration membrane filtration sterilization of 0.10 μ m, promptly get the liquid serum-free animal cell culture medium, refrigeration is subsequent use down at 2 ℃.

Embodiment 3

Table 7

Component	g/100L	Component	g/100L
				Calcium Chloride Powder Anhydrous	18	The L-tryptophane	1
Salzburg vitriol	0.0004	L-tyrosine	10
				Nine nitric hydrate iron	0.005	The L-Xie Ansuan	7
Presfersul	0.2	D-glucose	600
				Repone K	50	HEPES	360
Magnesium dichloride hexahydrate	18	Linolic acid	0.01
				Anhydrous magnesium sulfate	4	Thioctic Acid	0.07
Sodium-chlor	670	1,4-tetramethylenediamine dihydrochloride	0.8
				AMSP	7	Sodium.alpha.-ketopropionate	17
Sodium phosphate, dibasic	6	Vitamin H	0.002
				Zinc vitriol	0.2	The D-VA	0.2
Anhydrous potassium dihydrogenphosphate	2	Choline chloride 60	7
				The L-arginine monohydrochloride	70	Folic acid	0.2
The L-cystine hydrochloride	5	The i-inositol	7
				L-glutaminate	170	Vitamin PP	0.2
Glycocoll	5	Pyridoxal hydrochloride	0.5
				The L-L-Histidine hydrochloride	5	Pyridoxine hydrochloride	0.01
The L-Isoleucine	8	Vitamin G	0.03
				The L-leucine	8	Thiamine hydrochloride	0.4
L lysine HCL	10	Thymidine	0.04
				The L-methionine(Met)	0.5	Cobalamin	0.8
The L-phenylalanine(Phe)	4	The recombinant human insulin	0.7
				The L-Serine	3.5	Selenous acid	0.0004
The L-Threonine	3	Thanomin	0
				The L-L-Ala	1	Sunlover 10	180
The L-asparagine	10	Semen Pisi sativi protein	180
				The L-aspartic acid	0.5	Broad bean albumen	170
The L-cysteine hydrochloride	14	Rhizoma Solani tuber osi protein	70
				L-L-glutamic acid	0	Wheat bran albumen	160
The L-proline(Pro)	16	Rice protein	80
				Xanthoglobulin	0.5	Phenol red	1

Solid ingredient shown in the table 7 takes by weighing with ten thousand/analytical balance according to the consumption of 10 liters of final liquid nutrient mediums.Wherein,, be dissolved in the 1mol/L NaOH distilled water solution of 1000ml, obtain A solution vitamin H, xanthoglobulin, thymidine; Linolic acid is dissolved in 10ml 99% ethanolic soln, obtains B solution; Choline chloride 60, D-VA, folic acid, vitamin PP, pyridoxine hydrochloride, vitamin G, thiamine hydrochloride, cobalamin, pyridoxal hydrochloride, recombinant human insulin, selenous acid be dissolved in obtain C solution in the 1000ml zero(ppm) water; Merge A solution, B solution and C solution, in the gained mixing solutions, add glucose then, be settled to 10000ml with zero(ppm) water and shake up; Collect the gained mixed solution; Use the medium-sized freeze drier of FD-3A (Xi'an moral growth Instr Ltd.), according to this instrument specification sheets, at precooling temperature-40 ℃; Lyophilize is 30 hours under the maintenance vacuum tightness that slowly the heats up 10Pa, obtains lyophilized powder.With AMSP, Repone K, anhydrous magnesium sulfate, sodium-chlor, ADSP, anhydrous potassium dihydrogenphosphate, Calcium Chloride Powder Anhydrous in 105 ℃ of dryings 2 hours, with L-L-Ala, L-aspartic acid, L-L-glutamic acid, L-glutaminate, L-L-Histidine hydrochloride, L-arginine monohydrochloride, L-Isoleucine, L-leucine, L lysine HCL, L-methionine(Met), L-phenylalanine(Phe), L-proline(Pro), L-Serine, L-tryptophane, L-Threonine, L-Xie Ansuan, L-tyrosine, glycocoll, L-cystine hydrochloride, phenol red in 40 ℃ dry 1 hour down.With Magnesium dichloride hexahydrate, Presfersul, Zinc vitriol, Sodium.alpha.-ketopropionate, cupric sulfate pentahydrate, nine water iron nitrates, HEPES, 1,4-tetramethylenediamine dihydrochloride, i-inositol, thanomin, Sunlover 10, Semen Pisi sativi protein, broad bean albumen, Rhizoma Solani tuber osi protein, Wheat bran albumen, rice protein, L-cysteine hydrochloride, L-asparagine were put into the vacuum drying oven inner drying 20 hours.Above-mentioned all dried components and lyophilized powder are packed in the ball grinder, and ball milling 10 hours promptly gets serum-free animal cell culture medium dry powder of the present invention.Serum-free animal cell culture medium dry powder seal at normal temperatures keep in Dark Place subsequent use.

Serum-free animal cell culture medium dry powder 296.3368 grams with embodiment 3 makes are dissolved in the 8L ultrapure water, are settled to 10L with ultrapure water then.Use the bacteriological filtration membrane filtration sterilization of 0.15 μ m, promptly get the liquid serum-free animal cell culture medium, refrigeration is subsequent use down at 2 ℃.

Embodiment 4

Table 8

Component	g/100L	Component	g/100L
				Calcium Chloride Powder Anhydrous	20	The L-tryptophane	1.5
Salzburg vitriol	0.0008	L-tyrosine	2
				Nine nitric hydrate iron	0.008	The L-Xie Ansuan	5
Presfersul	0.3	D-glucose	700
				Repone K	40	HEPES	400
Magnesium dichloride hexahydrate	20	Linolic acid	0.05
				Anhydrous magnesium sulfate	2	Thioctic Acid	0.06
Sodium-chlor	700	1,4-tetramethylenediamine dihydrochloride	1
				AMSP	4	Sodium.alpha.-ketopropionate	20
Sodium phosphate, dibasic	8	Vitamin H	0.003
				Zinc vitriol	0.3	The D-VA	0.5
Anhydrous potassium dihydrogenphosphate	10	Choline chloride 60	9
				The L-arginine monohydrochloride	90	Folic acid	0.4
The L-cystine hydrochloride	10	The i-inositol	10
				L-glutaminate	200	Vitamin PP	0.4
Glycocoll	3	Pyridoxal hydrochloride	0.4
				The L-L-Histidine hydrochloride	4	Pyridoxine hydrochloride	0.05
The L-Isoleucine	10	Vitamin G	0.04
				The L-leucine	5	Thiamine hydrochloride	0.3
L lysine HCL	7	Thymidine	0.03
				The L-methionine(Met)	5	Cobalamin	0.4
The L-phenylalanine(Phe)	5	The recombinant human insulin	1
				The L-Serine	2	Selenous acid	0.0009
The L-Threonine	8	Thanomin	0.0005
				The L-L-Ala	0.4	Sunlover 10	140
The L-asparagine	12	Semen Pisi sativi protein	150
				The L-aspartic acid	2	Broad bean albumen	200
The L-cysteine hydrochloride	17	Rhizoma Solani tuber osi protein	90
				L-L-glutamic acid	2	Wheat bran albumen	170
The L-proline(Pro)	8	Rice protein	60
				Xanthoglobulin	0.5	Phenol red	1

Solid ingredient shown in the table 8 takes by weighing with ten thousand/analytical balance according to the consumption of 10 liters of final liquid nutrient mediums.Wherein, vitamin H, xanthoglobulin, thymidine are dissolved in the 1mol/L NaOH distilled water solution of 1000ml, obtain A solution; With the L-L-Ala; The L-aspartic acid; L-L-glutamic acid; L-glutaminate; The L-L-Histidine hydrochloride; L lysine HCL; The L-arginine monohydrochloride; The L-Isoleucine; The L-leucine; The L-methionine(Met); The L-phenylalanine(Phe); The L-proline(Pro); The L-Serine; The L-tryptophane; The L-Threonine; The L-Xie Ansuan; Choline chloride 60; The D-VA; Folic acid; Vitamin PP; Pyridoxine hydrochloride; Pyridoxal hydrochloride; Vitamin G; Thiamine hydrochloride; Cobalamin; Pyridoxal hydrochloride; The recombinant human insulin; Selenous acid is dissolved in and obtains B solution in the 3000ml zero(ppm) water; The ethanol that linolic acid is dissolved in 10ml99% obtains C solution; It is to obtain D solution in 1 the hydrochloric acid distilled water solution that L-tyrosine is scattered in the pH value; Merge A solution, B solution, C solution and D solution; In the gained mixing solutions, add glucose, AMSP, Repone K, anhydrous magnesium sulfate, Calcium Chloride Powder Anhydrous, sodium-chlor, ADSP, anhydrous potassium dihydrogenphosphate, glycocoll, L-cystine hydrochloride, L-cysteine hydrochloride, L-asparagine, Presfersul, Zinc vitriol, Sodium.alpha.-ketopropionate, cupric sulfate pentahydrate, nine water iron nitrates, HEPES, 1 then; 4-tetramethylenediamine dihydrochloride, i-inositol, thanomin, Sunlover 10, Semen Pisi sativi protein, broad bean albumen, Rhizoma Solani tuber osi protein, Wheat bran albumen, rice protein are settled to 10L with zero(ppm) water and shake up.Promptly get the liquid serum-free animal cell culture medium, refrigeration is subsequent use down at 2 ℃.

Embodiment 5

Table 9

Component	g/100L	Component	g/100L
				Calcium Chloride Powder Anhydrous	20	The L-tryptophane	0.5
Salzburg vitriol	0.0001	L-tyrosine	7
				Nine nitric hydrate iron	0	The L-Xie Ansuan	3
Presfersul	0.2	D-glucose	550
				Repone K	30	HEPES	400
Magnesium dichloride hexahydrate	1	Linolic acid	0.03
				Anhydrous magnesium sulfate	6	Thioctic Acid	0.05
Sodium-chlor	600	1,4-tetramethylenediamine dihydrochloride	0.7
				AMSP	5	Sodium.alpha.-ketopropionate	20
Sodium phosphate, dibasic	6	Vitamin H	0.001
				Zinc vitriol	0.04	The D-VA	0.4
Anhydrous potassium dihydrogenphosphate	8	Choline chloride 60	5
				The L-arginine monohydrochloride	20	Folic acid	0.4
The L-cystine hydrochloride	4	The i-inositol	2
				L-glutaminate	150	Vitamin PP	0.1
Glycocoll	6	Pyridoxal hydrochloride	0.5
				The L-L-Histidine hydrochloride	2	Pyridoxine hydrochloride	0.01
The L-Isoleucine	4	Vitamin G	0.02
				The L-leucine	9	Thiamine hydrochloride	0.3
L lysine HCL	5	Thymidine	0.01
				The L-methionine(Met)	1	Cobalamin	0.7
The L-phenylalanine(Phe)	2	The recombinant human insulin	0.2
				The L-Serine	1.5	Selenous acid	0.0001
The L-Threonine	8	Thanomin	0.0005
				The L-L-Ala	0.2	Sunlover 10	170
The L-asparagine	15	Semen Pisi sativi protein	120
				The L-aspartic acid	0.5	Broad bean albumen	170
The L-cysteine hydrochloride	10	Rhizoma Solani tuber osi protein	0
				L-L-glutamic acid	1.5	Wheat bran albumen	100
The L-proline(Pro)	8	Rice protein	50

With the consumption of the solid ingredient shown in the table 9 according to 100 liters of final liquid nutrient mediums; After accurately weighing mixes one by one with ten thousand/balance, add 50 liters of zero(ppm) water again, with the stirring of Mei Ying Pu, the Shanghai 85-2 of instrument Manufacturing Co., Ltd type whisking appliance after 2 hours; Obtain homogeneous dispersion; Use the medium-sized freeze drier of FD-3A (Xi'an moral growth Instr Ltd.), according to this instrument specification sheets, at precooling temperature-45 ℃; Lyophilize is 50 hours under the slow interior vacuum tightness 9Pa of maintenance instrument that heats up, and obtains serum-free animal cell culture medium dry powder of the present invention.Serum-free animal cell culture medium dry powder seal at normal temperatures keep in Dark Place subsequent use.

With the above-mentioned serum-free animal cell culture medium dry powder of the present invention that makes 2524.862 grams, be dissolved in 30 ℃ of 100L ultrapure waters, gained solution uses the bacteriological filtration membrane filtration sterilization of 0.20 μ m, promptly gets the liquid serum-free animal cell culture medium, and refrigeration is subsequent use down at 2 ℃.

Embodiment 6

Table 10

Component	g/100L	Component	g/100L
				Calcium Chloride Powder Anhydrous	10	The L-tryptophane	2
Salzburg vitriol	0	L-tyrosine	10
				Nine nitric hydrate iron	0.01	The L-Xie Ansuan	10
Presfersul	0.1	D-glucose	700
				Repone K	20	HEPES	330
Magnesium dichloride hexahydrate	10	Linolic acid	0.05
				Anhydrous magnesium sulfate	4	Thioctic Acid	0.08
Sodium-chlor	620	1,4-tetramethylenediamine dihydrochloride	0.02
				AMSP	7	Sodium.alpha.-ketopropionate	10
Sodium phosphate, dibasic	10	Vitamin H	0.005
				Zinc vitriol	0.01	The D-VA	0.2
Anhydrous potassium dihydrogenphosphate	5	Choline chloride 60	10
				The L-arginine monohydrochloride	80	Folic acid	0.2
The L-cystine hydrochloride	7	The i-inositol	10
				L-glutaminate	50	Vitamin PP	0.5
Glycocoll	8	Pyridoxal hydrochloride	0.3
				The L-L-Histidine hydrochloride	1	Pyridoxine hydrochloride	0.05
The L-Isoleucine	10	Vitamin G	0.03
				The L-leucine	6	Thiamine hydrochloride	0.4
L lysine HCL	12	Thymidine	0.05
				The L-methionine(Met)	3.5	Cobalamin	0.5
The L-phenylalanine(Phe)	6	The recombinant human insulin	1
				The L-Serine	2.5	Selenous acid	0.0009
The L-Threonine	3	Thanomin	0.0001
				The L-L-Ala	1	Sunlover 10	140
The L-asparagine	6	Semen Pisi sativi protein	200
				The L-aspartic acid	2	Broad bean albumen	150
The L-cysteine hydrochloride	20	Rhizoma Solani tuber osi protein	50
				L-L-glutamic acid	2	Wheat bran albumen	40
The L-proline(Pro)	10	Rice protein	100
				Xanthoglobulin	0.3

Solid ingredient shown in the table 10 takes by weighing with ten thousand/analytical balance according to the consumption of 100 liters of final liquid nutrient mediums.Wherein, vitamin H, xanthoglobulin, thymidine are dissolved in the 1mol/L NaOH distilled water solution of 1000ml, obtain A solution; With the L-L-Ala; The L-aspartic acid; L-L-glutamic acid; L-glutaminate; The L-L-Histidine hydrochloride; L lysine HCL; The L-arginine monohydrochloride; The L-Isoleucine; The L-leucine; The L-methionine(Met); The L-phenylalanine(Phe); The L-proline(Pro); The L-Serine; The L-tryptophane; The L-Threonine; The L-Xie Ansuan; Choline chloride 60; The D-VA; Folic acid; Vitamin PP; Pyridoxine hydrochloride; Vitamin G; Thiamine hydrochloride; Cobalamin; Pyridoxal hydrochloride; The recombinant human insulin; Selenous acid is dissolved in and obtains B solution in the 3000ml zero(ppm) water; The ethanol that linolic acid is dissolved in 10ml 99% obtains C solution; It is to obtain D solution in 1 the hydrochloric acid distilled water solution that L-tyrosine is scattered in the pH value; Merge A solution, B solution, C solution and D solution; In the gained mixing solutions, add glucose, AMSP, Repone K, anhydrous magnesium sulfate, Calcium Chloride Powder Anhydrous, sodium-chlor, ADSP, anhydrous potassium dihydrogenphosphate, glycocoll, L-cystine hydrochloride, L-cysteine hydrochloride, L-asparagine, Presfersul, Zinc vitriol, Sodium.alpha.-ketopropionate, cupric sulfate pentahydrate, nine water iron nitrates, HEPES, 1 then; 4-tetramethylenediamine dihydrochloride, i-inositol, thanomin, Sunlover 10, Semen Pisi sativi protein, broad bean albumen, Rhizoma Solani tuber osi protein, Wheat bran albumen, rice protein; Being settled to 10000ml with zero(ppm) water shakes up; Collect the gained mixed solution; Use the medium-sized freeze drier of FD-3A (Xi'an moral growth Instr Ltd.), according to this instrument specification sheets, at precooling temperature-50 ℃; Lyophilize is 40 hours under the maintenance vacuum tightness that slowly the heats up 10Pa, promptly obtains serum-free animal cell culture medium dry powder.Serum-free animal cell culture medium dry powder seal at normal temperatures keep in Dark Place subsequent use.

With the above-mentioned Zooblast culture medium dry powder that makes 2681.806 grams, be dissolved in the 80L ultrapure water, be settled to 100L with ultrapure water then.Use the bacteriological filtration membrane filtration sterilization of 0.10 μ m, promptly get the liquid serum-free animal cell culture medium, refrigeration is subsequent use down at 2 ℃.

Embodiment 7

Table 11

Component	g/100L	Component	g/100L
				Calcium Chloride Powder Anhydrous	17	The L-tryptophane	1
Salzburg vitriol	0.001	L-tyrosine	2
				Nine nitric hydrate iron	0.008	The L-Xie Ansuan	5
Presfersul	0.5	D-glucose	600
				Repone K	50	HEPES	370
Magnesium dichloride hexahydrate	6	Linolic acid	0.02
				Anhydrous magnesium sulfate	8	Thioctic Acid	0.1
Sodium-chlor	670	1,4-tetramethylenediamine dihydrochloride	0.9
				AMSP	8	Sodium.alpha.-ketopropionate	11
Sodium phosphate, dibasic	7	Vitamin H	0.002
				Zinc vitriol	0.02	The D-VA	0.1
Anhydrous potassium dihydrogenphosphate	2	Choline chloride 60	6
				The L-arginine monohydrochloride	30	Folic acid	0.1
The L-cystine hydrochloride	3	The i-inositol	3
				L-glutaminate	120	Vitamin PP	0.2
Glycocoll	2	Pyridoxal hydrochloride	0.2
				The L-L-Histidine hydrochloride	3	Pyridoxine hydrochloride	0.04
The L-Isoleucine	7	Vitamin G	0.01
				The L-leucine	4	Thiamine hydrochloride	0.1
L lysine HCL	9	Thymidine	0.04
				The L-methionine(Met)	0.5	Cobalamin	1
The L-phenylalanine(Phe)	5	The recombinant human insulin	0.5
				The L-Serine	3	Selenous acid	0.001
The L-Threonine	7	Thanomin	0.0002
				The L-L-Ala	0.6	Sunlover 10	100
The L-asparagine	12	Semen Pisi sativi protein	140
				The L-aspartic acid	1	Broad bean albumen	180
The L-cysteine hydrochloride	13	Rhizoma Solani tuber osi protein	25
				L-L-glutamic acid	0.5	Wheat bran albumen	60
The L-proline(Pro)	17	Rice protein	60
				Xanthoglobulin	0.5	Phenol red	1

Solid ingredient shown in the table 11 takes by weighing with ten thousand/analytical balance according to the consumption of 10 liters of final liquid nutrient mediums.Wherein,, be dissolved in the 1mol/L NaOH distilled water solution of 1000ml, obtain A solution vitamin H, xanthoglobulin, thymidine; Linolic acid is dissolved in 10ml 99% ethanolic soln, obtains B solution; Choline chloride 60, D-VA, folic acid, vitamin PP, pyridoxine hydrochloride, vitamin G, thiamine hydrochloride, cobalamin, pyridoxal hydrochloride, recombinant human insulin, selenous acid be dissolved in obtain C solution in the 1000ml zero(ppm) water; Merge A solution, B solution and C solution, in the gained mixing solutions, add glucose then, be settled to 10000ml with zero(ppm) water and shake up; Collect the gained mixed solution; Use the medium-sized freeze drier of FD-3A (Xi'an moral growth Instr Ltd.), according to this instrument specification sheets, at precooling temperature-40 ℃; Lyophilize is 30 hours under the maintenance vacuum tightness that slowly the heats up 10Pa, obtains lyophilized powder.With AMSP, Repone K, anhydrous magnesium sulfate, sodium-chlor, ADSP, anhydrous potassium dihydrogenphosphate, Calcium Chloride Powder Anhydrous in 105 ℃ of dryings 2 hours, with L-L-Ala, L-aspartic acid, L-L-glutamic acid, L-glutaminate, L-L-Histidine hydrochloride, L-arginine monohydrochloride, L-Isoleucine, L-leucine, L lysine HCL, L-methionine(Met), L-phenylalanine(Phe), L-proline(Pro), L-Serine, L-tryptophane, L-Threonine, L-Xie Ansuan, L-tyrosine, glycocoll, L-cystine hydrochloride, phenol red in 40 ℃ dry 1 hour down.With Magnesium dichloride hexahydrate, Presfersul, Zinc vitriol, Sodium.alpha.-ketopropionate, cupric sulfate pentahydrate, nine water iron nitrates, HEPES, 1,4-tetramethylenediamine dihydrochloride, i-inositol, thanomin, Sunlover 10, Semen Pisi sativi protein, broad bean albumen, Rhizoma Solani tuber osi protein, Wheat bran albumen, rice protein, L-cysteine hydrochloride, L-asparagine were put into the vacuum drying oven inner drying 20 hours.Above-mentioned all dried components and lyophilized powder are packed in the ball grinder, and ball milling 10 hours promptly gets serum-free animal cell culture medium dry powder of the present invention.Serum-free animal cell culture medium dry powder seal at normal temperatures keep in Dark Place subsequent use.

Serum-free animal cell culture medium dry powder 257.3942 grams with embodiment 7 makes are dissolved in the 8L ultrapure water, are settled to 10L with ultrapure water then.Use the bacteriological filtration membrane filtration sterilization of 0.15 μ m, promptly get the liquid serum-free animal cell culture medium, refrigeration is subsequent use down at 2 ℃.

Embodiment 8

Table 12

Component	g/100L	Component	g/100L
				Calcium Chloride Powder Anhydrous	11	The L-tryptophane	1.5
Salzburg vitriol	0.0009	L-tyrosine	3
				Nine nitric hydrate iron	0.006	The L-Xie Ansuan	9
Presfersul	0.3	D-glucose	500
				Repone K	10	HEPES	300
Magnesium dichloride hexahydrate	2	Linolic acid	0.01
				Anhydrous magnesium sulfate	2	Thioctic Acid	0.06
Sodium-chlor	700	1,4-tetramethylenediamine dihydrochloride	1
				AMSP	2	Sodium.alpha.-ketopropionate	15
Sodium phosphate, dibasic	8	Vitamin H	0.003
				Zinc vitriol	0.05	The D-VA	0.5
Anhydrous potassium dihydrogenphosphate	10	Choline chloride 60	7
				The L-arginine monohydrochloride	50	Folic acid	0.5
The L-cystine hydrochloride	10	The i-inositol	7
				L-glutaminate	60	Vitamin PP	0.4
Glycocoll	7	Pyridoxal hydrochloride	0.1
				The L-L-Histidine hydrochloride	5	Pyridoxine hydrochloride	0.02
The L-Isoleucine	9	Vitamin G	0.05
				The L-leucine	10	Thiamine hydrochloride	0.5
L lysine HCL	8	Thymidine	0.03
				The L-methionine(Met)	5	Cobalamin	0.1
The L-phenylalanine(Phe)	4	The recombinant human insulin	0.9
				The L-Serine	5	Selenous acid	0.0004
The L-Threonine	5	Thanomin	0
				The L-L-Ala	0.3	Sunlover 10	200
The L-asparagine	8	Semen Pisi sativi protein	190
				The L-aspartic acid	1.5	Broad bean albumen	120
The L-cysteine hydrochloride	18	Rhizoma Solani tuber osi protein	45
				L-L-glutamic acid	0	Wheat bran albumen	0
The L-proline(Pro)	20	Rice protein	90
				Xanthoglobulin	0.5	Phenol red	1

Solid ingredient shown in the table 12 takes by weighing with ten thousand/analytical balance according to the consumption of 10 liters of final liquid nutrient mediums.Wherein, vitamin H, xanthoglobulin, thymidine are dissolved in the 1mol/L NaOH distilled water solution of 1000ml, obtain A solution; With the L-L-Ala; The L-aspartic acid; L-L-glutamic acid; L-glutaminate; The L-L-Histidine hydrochloride; L lysine HCL; The L-arginine monohydrochloride; The L-Isoleucine; The L-leucine; The L-methionine(Met); The L-phenylalanine(Phe); The L-proline(Pro); The L-Serine; The L-tryptophane; The L-Threonine; The L-Xie Ansuan; Choline chloride 60; The D-VA; Folic acid; Vitamin PP; Pyridoxine hydrochloride; Pyridoxal hydrochloride; Vitamin G; Thiamine hydrochloride; Cobalamin; Pyridoxal hydrochloride; The recombinant human insulin; Selenous acid is dissolved in and obtains B solution in the 3000ml zero(ppm) water; The ethanol that linolic acid is dissolved in 10ml99% obtains C solution; It is to obtain D solution in 1 the hydrochloric acid distilled water solution that L-tyrosine is scattered in the pH value; Merge A solution, B solution, C solution and D solution; In the gained mixing solutions, add glucose, AMSP, Repone K, anhydrous magnesium sulfate, Calcium Chloride Powder Anhydrous, sodium-chlor, ADSP, anhydrous potassium dihydrogenphosphate, glycocoll, L-cystine hydrochloride, L-cysteine hydrochloride, L-asparagine, Presfersul, Zinc vitriol, Sodium.alpha.-ketopropionate, cupric sulfate pentahydrate, nine water iron nitrates, HEPES, 1 then; 4-tetramethylenediamine dihydrochloride, i-inositol, thanomin, Sunlover 10, Semen Pisi sativi protein, broad bean albumen, Rhizoma Solani tuber osi protein, Wheat bran albumen, rice protein are settled to 10L with zero(ppm) water and shake up.Promptly get the liquid serum-free animal cell culture medium, refrigeration is subsequent use down at 2 ℃.

Vegetable-protein makes according to following method described in the above embodiment 1-8, and the conditional parameter of concrete preparation is seen table 13.Said method comprising the steps of:

A) milling soya seeds, pea, broad bean, yam, Wheat bran or rice plants raw material are to 0.1-3mm ³Particle;

C) keep 5-20min to stop the described enzymolysis of step b) at 80-100 ℃;

Table 13

Embodiment

1

Embodiment 2

Embodiment 3

Embodiment 4

Embodiment 5

Embodiment 6

Embodiment 7

Embodiment 8

Particle diameter (mm ³)

0.1-3

0.1-2

0.1-1

0.5-3

0.7-2

0.4-2.5

1-3

0.8-3

Enzyme

Papoid

Sumizyme MP

Neutral protease

Trypsinase

Bromeline

Papoid

Trypsinase

Enzyme (units/gram) alive

10 ⁵

10 ⁶

10 ⁷

10 ⁵

10 ⁷

10 ⁶

10 ⁵

Enzymolysis time (h)

5

2

1

3

4

6

7

8

Final temperature (℃)/time (min)

80/15

95/20

90/17

100/5

93/8

85/19

96/12

89/13

Centrifugal rotational speed/time (min)

4000/15

3000/20

4500/17

8000/5

7500/8

5000/19

6000/12

6500/13

Film sees through molecular weight ranges

10000- 20000

10000- 15000

10000- 20000

15000- 20000

10000- 20000

Comparative Examples 1

This Comparative Examples is used to explain existing liquid serum-free animal cell culture medium and preparation method thereof.

Table 14 is prescriptions of the embodiment 4 record tables 2 of CN 1318577C, and according to the serum-free animal cell culture medium of this formulation liquid prior art, refrigeration is subsequent use down at 2 ℃.

Table 14

Component	g/L
		Minimum medium (DMEM/HAM ' s F12)	11.5
Soy peptone	2.5
		The L Stimulina	0.36
Xitix	0.0035
		NaHCO ₃	2.00
Thanomin	0.0015
		Sodium Selenite	8.6μg/l
Synperonic?F68	0.25

Comparative Examples 2

According to the prescription that the table 13 (the application's ordering is table 15) of WO 02/29084 is put down in writing, the serum-free animal cell culture medium of preparation liquid prior art, refrigeration is subsequent use down at 2 ℃.

Table 15

Component	Content (mg/l)	Component	Content (mg/l)
				Sodium-chlor	6122	The L-methionine(Met)	137.24
Repone K	311.8	The L-phenylalanine(Phe)	155.48
				One hypophosphite monohydrate sodium dihydrogen	62.5	The L-proline(Pro)	17.25
Sodium hydrogencarbonate	0	The L-Serine	266.25
				ADSP	71.02	The L-Threonine	173.45
Seven hypophosphite monohydrate disodium hydrogens	0	The L-tryptophane	39.02
				Magnesium Chloride Anhydrous	28.64	L-tyrosine disodium duohydrate (L-tyrosine disodium dihydrate)	55.79
Magnesium dichloride hexahydrate	0	The L-Xie Ansuan	177.85
				Anhydrous magnesium sulfate	48.84	L-Gelucystine dihydrochloride	31.29
Bitter salt	0	Xanthoglobulin sodium (Sodium hypoxanthine)	2.39
				Calcium Chloride Powder Anhydrous	116.6	Putrescine dihydrochloride (Putrescine dihydrochloride)	0.081
Salzburg vitriol	0.0013	Sodium.alpha.-ketopropionate	220
				Presfersul	0.147	The D-vitamin H	0.1313
Nine nitric hydrate iron	0.05	The D-VA	4.08
				Ironic citrate	0.4	Folic acid	4.65
Zinc vitriol	0.432	The i-inositol	39.1
				Dextrose anhydrous	4501	Vitamin PP	3.085
Linolic acid	1.189	Choline chloride 60	29.32
				Regular Insulin	5	Benadon	0.117
DL 68 Thioctic Acids	0.473	Vitamin G	0.219
				The L-L-Ala	4.45	Thiamine hydrochloride	2.67
Chlorination L-l-arginine (L-arginine chloride)	547.8	Thymidine	0.365
				L-asparagine monohydrate (L-asparagine monohydrate)	407.5	Cobalamin	2.68
The L-aspartic acid	6.65	Pyridoxal hydrochloride	6
				L-halfcystine hydrochloric acid monohydrate (L-cysteine hydrochloride monohydrate)	117.65	Triptide	2.5
L-L-glutamic acid	251.35	Sodium Selenite	0.02175

Glycocoll	18.75	The L-xitix	27.5
				L-Histidine hydrochloride monohydrate	211.48	Pluronic F68 (Pluronic F68)	1000
The L-Isoleucine	54.47	Vitamin K	5
				The L-leucine	179.05	VISOSE D70	0
L lysine HCL	231.25	?HY-SOY	500

EXPERIMENTAL EXAMPLE 1

Under aseptic condition, respectively 50ml aforesaid liquid serum-free animal cell culture medium is transferred to the 250ml cell cultures with the sterilization pipettor and shake in the bottle, inoculation Chinese hamster ovary celI suspension, inoculum density is 5 * 10 ⁵Cell/ml, 20 bottles of every kind of culture medium inoculateds.Rotating speed with 120 rev/mins carries out suspension culture under 37 ℃ of temperature condition, 95% humidity condition and 5% carbon dioxide conditions then.With the cell density of counting method of blood cell record unit fate, measure viable cell density with mtt assay.Cultivation results is seen table 16 and Fig. 7 A.72 hours cell photo of embodiment 1 culture medium culturing is seen Figure 1A; 72 hours cell photo of Comparative Examples 1 culture medium culturing is seen Fig. 2 A; 72 hours cell photo of Comparative Examples 2 culture medium culturing is seen Fig. 3 A; 72 hours cell photo of embodiment 2 thin culture medium culturing is seen Fig. 4 A; 48 hours cell photo of embodiment 3 culture medium culturing is seen Fig. 5 A; 48 hours cell photo of embodiment 4 culture medium culturing is seen Fig. 6 A.

Table 16

Can find out that from the result of table 16 when serum-free animal cell culture medium dry powder of the present invention or liquid serum free medium were used to cultivate Chinese hamster ovary celI, the cell culture density that can reach improved greatly.

EXPERIMENTAL EXAMPLE 2

Under aseptic condition, respectively 50ml aforesaid liquid serum-free animal cell culture medium to be transferred to the 250ml cell cultures with the sterilization pipettor and shake in the bottle, the Chinese hamster ovary celI suspension of IgG antibody is expressed in inoculation, and inoculum density is 5 * 10 ⁵Cell/ml, 20 bottles of every kind of culture medium inoculateds.Rotating speed with 90 rev/mins carried out suspension culture 3-4 days under 37 ℃ of temperature condition, 95% humidity condition and 5 volume % carbon dioxide conditions then.Repeat said process and carry out five batches cultivation.After cultivating end, it is centrifugal under 2000g to get cell culture, separation and Culture thing supernatant.With Protein A chromatography separate targets antibody, measure purity with SDS-PAGE protein electrophoresis method.

Measure respectively with the ELISA method and to cultivate after 6 days the concentration of antibody in the cell conditioned medium liquid, the result is shown in table 17.

Table 17

Substratum

Embodiment

1

Embodiment 2

Embodiment 3

Embodiment 4

Comparative Examples 1

Comparative Examples 2

20 bottles of average antibody concentration (μ g/ml)

?148

139

?137

?143

109

114

Five batches of AC standard deviations

?±8

±5

?±6

?±7

±11

±10

Purity (%)

?97

96

?95

?98

96

97

Can find out from table 17, because the composition that substratum of the present invention adds confirms that make by batch otherness of culturing cell to reduce greatly, security is better.Substratum of the present invention in addition helps the isolated cell derived product, and cost is reduced.

EXPERIMENTAL EXAMPLE 3

Under aseptic condition, respectively 50ml aforesaid liquid serum-free animal cell culture medium is transferred to the 250ml cell cultures with the sterilization pipettor and shake in the bottle, inoculation BHK21 cell suspension, inoculum density is 8 * 10 ⁵Cell/ml, 20 bottles of every kind of culture medium inoculateds.Rotating speed with 120 rev/mins carries out suspension culture under 37 ℃ of temperature condition, 95% humidity condition and 5 volume % carbon dioxide conditions then.With the cell density of counting method of blood cell record unit fate, measure cell viability (being the per-cent that viable cell accounts for TCS) with mtt assay.。Cultivation results is seen table 18 and Fig. 7 B.72 hours cell photo of embodiment 5 culture medium culturing is seen Figure 1B; 72 hours cell photo of Comparative Examples 1 culture medium culturing is seen Fig. 2 B; 72 hours cell photo of Comparative Examples 2 culture medium culturing is seen Fig. 3 B; 72 hours cell photo of embodiment 6 thin culture medium culturing is seen Fig. 4 B; 48 hours cell photo of embodiment 7 culture medium culturing is seen Fig. 5 B; 48 hours cell photo of embodiment 8 culture medium culturing is seen Fig. 6 B.

Table 18

Can find out that from the result of table 18 when serum-free animal cell culture medium dry powder of the present invention or liquid serum free medium were used to cultivate the BHK21 cell, the cell culture density that can reach improved greatly.

EXPERIMENTAL EXAMPLE 4

Cell culture medium: press formulated BHK21 serum-free cell culture medium among embodiment 5-8 and the Comparative Examples 1-2.

Bio-reactor: 650L stirring type bioreactor (Beijing Qingdatianyi Bioisystech Co., Ltd)

Volume of culture: 200L

Inoculum density: 6 * 10 ⁵

Cell culture condition: temperature is that 37 ℃, pH value are 7.3, dissolved oxygen 60%.

Cultivated 7 days, cell density is 5 * 10 ⁶During cell/ml the left and right sides, by virus infection plural number MOI 0.1 amount inoculation rabies virus.

Virus is kept liquid: except that not comprising broad bean albumen, Rhizoma Solani tuber osi protein, Wheat bran albumen and rice protein, other are identical with cell culture medium.

The virus culture condition: temperature is that 34 ℃, pH value are 7.6, dissolved oxygen 55%.

Cultivate after 48 hours, keep liquid with the speed fresh virus that perfusion is sterilized from the containers for culturing organisms bottom in 100-200 liter/sky, and collect virus from containers for culturing organisms top near the substratum liquid level.Continous pouring was cultivated 10 days, gathered in the crops 1400 liters of viral solution altogether.With assay method method in the mouse brain in Chinese Pharmacopoeia (2005 editions) Antirabic Vaccine's rules, from second day, to the rabies virus titre (LD of the reactor drum sample got every day ₅₀) measure.The result is shown in table 19.

Table 19

Virus titer (LD ₅₀)	The 2nd day	The 3rd day	The 4th day	The 5th day	The 6th day	The 7th day	The 8th day	The 9th day	The 10th day
										Embodiment 5	3.2	4.6	6.1	6.5	6.8	7.4	7.8	7.5	7.3
Embodiment 6	/	3.5	4.9	5.6	6.7	7.3	7.7	7.9	7.6
										Embodiment 7	3.4	4.5	5.3	6.8	7.8	8.1	7.8	7.7	7.2
Embodiment 8	/	3.5	4.8	5.6	6.7	7.8	7.4	7.2	7.0
										Comparative Examples 1	/	3.4	4.7	5.4	6.3	/	/	/	/
Comparative Examples 2	/	3.2	4.9	5.6	6.5	/	/	/	/

Wherein "/" expression virus titer is low excessively, can not measure effective titre.

Can find out from the result of table 19; When serum-free animal cell culture medium dry powder of the present invention or liquid serum free medium are used for cultivating the BHK21 cell and are used to produce rabies virus vaccine process; The virus titer that can gather in the crops improves greatly, and can produce the cycle stretch-out of effective virus titer.

EXPERIMENTAL EXAMPLE 5

Bio-reactor: 100L stirring type bioreactor.

Volume of culture: 50L

Inoculum density: 3 * 10 ⁵

Cell culture condition: temperature is that 36 ℃, pH value are 7.2, dissolved oxygen 50%.

Cultivated 5 days, cell density is 1 * 10 ⁶During cell/ml the left and right sides, by virus infection plural number MOI 0.05 amount inoculation foot and mouth disease virus.

The virus culture condition: temperature is that 35 ℃, pH value are 7.4, dissolved oxygen 50%.

Cultivate after 48 hours, keep liquid with the fresh virus of 40 liters/day speed perfusion sterilization, and collect virus from containers for culturing organisms top near the substratum liquid level from the containers for culturing organisms bottom.Continous pouring was cultivated 15 days, gathered in the crops 450 liters of viral solution altogether.With suckling mouse medium lethal dose(LD&-{50}) measuring method, from 6h, to the foot and mouth disease virus titre (LD of the reactor drum sample that every 6h or 3h got ₅₀) measure.The result is shown in table 20.

Table 20

Virus titer (LD ₅₀)	6h	12h	18h	21h	24h
						Embodiment 5	6.5	7.35	8.0	7.75	7.5
Embodiment 6	6.75	7.35	8.0	7.25	7.0
						Embodiment 7	6.5	7.5	7.75	7.5	7.25
Embodiment 8	7.0	7.5	8.25	8.0	7.75
						Comparative Examples 1	6.0	7.0	7.25	7.0	6.5
Comparative Examples 2	6.25	7.25	7.5	7.25	6.75

Can find out that from the result of table 20 when serum-free animal cell culture medium dry powder of the present invention or liquid serum free medium were used for cultivating the BHK21 cell and are used to produce the foot and mouth disease virus vaccine process, the virus titer that can gather in the crops improved greatly.

Claims

1. serum-free animal cell culture medium dry powder; Said culture medium dry powder is dispersed in the substratum solvent and forms liquid nutrient medium; Said culture medium dry powder comprises minimum medium dry powder; Wherein, Said minimum medium is selected from one or more in BME cell culture medium, DMEM cell culture medium, DMEM/F12 cell culture medium, Fischer ' s cell culture medium, IMDM cell culture medium, 199 cell culture mediums, MEM cell culture medium, F10 cell culture medium, F12 cell culture medium and 1640 cell culture mediums; And wherein; Said substratum solvent is selected from one or more in zero(ppm) water, deionized water, water for injection, redistilled water and the ultrapure water; It is characterized in that, be benchmark with the volume of said substratum solvent, and said culture medium dry powder also comprises the Sunlover 10 of 100-200g/100L, the Semen Pisi sativi protein of 100-200g/100L, the broad bean albumen of 100-200g/100L, the Rhizoma Solani tuber osi protein of 0-100g/100L, the Wheat bran albumen of 0-200g/100L and the rice protein of 50-100g/100L.

2. serum-free animal cell culture medium dry powder according to claim 1; Wherein, Volume with said substratum solvent is a benchmark, and said culture medium dry powder also comprises the Sunlover 10 of 150-180g/100L, the Semen Pisi sativi protein of 150-180g/100L, the broad bean albumen of 150-180g/100L, the Rhizoma Solani tuber osi protein of 50-80g/100L, the Wheat bran albumen of 150-180g/100L and the rice protein of 50-80g/100L.

3. serum-free animal cell culture medium dry powder according to claim 2; Wherein, Volume with said substratum solvent is a benchmark, and said culture medium dry powder also comprises the Sunlover 10 of 170-180g/100L, the Semen Pisi sativi protein of 170-180g/100L, the broad bean albumen of 170-180g/100L, the Rhizoma Solani tuber osi protein of 50-60g/100L, the Wheat bran albumen of 150-160g/100L, the rice protein of 50-60g/100L.

4. according to any described serum-free animal cell culture medium dry powder among the claim 1-3, wherein, said Sunlover 10, Semen Pisi sativi protein, broad bean albumen, Rhizoma Solani tuber osi protein, Wheat bran albumen and rice protein prepare through following method:

C) keep 5-20min to stop the described enzymolysis of step b) at 80-100 ℃;

5. serum-free animal cell culture medium dry powder according to claim 4, wherein, said Sunlover 10, Semen Pisi sativi protein, broad bean albumen, Rhizoma Solani tuber osi protein, Wheat bran albumen and rice protein prepare through following method:

C) keep 5-10min to stop the described enzymolysis of step b) at 80-90 ℃;

6. serum-free animal cell culture medium dry powder according to claim 1 wherein, is benchmark with the volume of said substratum solvent, and said serum-free animal cell culture medium dry powder comprises the component shown in the following table 1:

Table 1

Component g/100L Component g/100L Calcium Chloride Powder Anhydrous 10-20 The L-tryptophane 0.5-2 Salzburg vitriol 0-0.001 L-tyrosine 2-10 Nine nitric hydrate iron 0-0.01 The L-Xie Ansuan 3-10 Presfersul 0.1-0.5 D-glucose 500-700 Repone K 10-50 HEPES 300-400 Magnesium dichloride hexahydrate 1-20 Linolic acid 0.01-0.05 Anhydrous magnesium sulfate 2-8 Thioctic Acid 0.05-0.1 Sodium-chlor 600-700 1,4-tetramethylenediamine dihydrochloride 0.02-1 AMSP 2-8 Sodium.alpha.-ketopropionate 10-20 Sodium phosphate, dibasic 6-10 Vitamin H 0.001-0.005 Zinc vitriol 0.01-0.5 The D-VA 0.1-0.5 Anhydrous potassium dihydrogenphosphate 2-10 Choline chloride 60 5-10 The L-arginine monohydrochloride 20-100 Folic acid 0.1-0.5 The L-cystine hydrochloride 3-10 The i-inositol 2-10 L-glutaminate 50-200 Vitamin PP 0.1-0.5 Glycocoll 2-8 Pyridoxal hydrochloride 0.1-0.5 The L-L-Histidine hydrochloride 1-5 Pyridoxine hydrochloride 0.01-0.05 The L-Isoleucine 4-10 Vitamin G 0.01-0.05 The L-leucine 4-10 Thiamine hydrochloride 0.1-0.5 L lysine HCL 5-12 Thymidine 0.01-0.05 The L-methionine(Met) 0.5-5 Cobalamin 0.1-1 The L-phenylalanine(Phe) 2-6 The recombinant human insulin 0.2-1 The L-Serine 1.5-5 Selenous acid 0.0001-0.001 The L-Threonine 3-8 Thanomin 0-0.0005 The L-L-Ala 0.2-1 Sunlover 10 100-200 The L-asparagine 6-15 Semen Pisi sativi protein 100-200 The L-aspartic acid 0.5-2 Broad bean albumen 100-200 The L-cysteine hydrochloride 10-20 Rhizoma Solani tuber osi protein 0-100 L-L-glutamic acid 0-2 Wheat bran albumen 0-200 The L-proline(Pro) 8-20 Rice protein 50-100

。

7. serum-free animal cell culture medium dry powder according to claim 6 wherein, is benchmark with the volume of said substratum solvent, and said serum-free animal cell culture medium dry powder also comprises xanthoglobulin and/or 0.1-2g/100L phenol red of 0.1-1g/100L.

8. serum-free animal cell culture medium dry powder according to claim 6 wherein, is benchmark with the volume of said substratum solvent, and said serum-free animal cell culture medium dry powder comprises the component shown in the following table 2:

Table 2

。

9. serum-free animal cell culture medium dry powder according to claim 6 wherein, is benchmark with the volume of said substratum solvent, and said serum-free animal cell culture medium dry powder comprises the component shown in the following table 3:

Table 3

Wherein, said serum-free animal cell culture medium dry powder is for cultivating the culture medium dry powder of Chinese hamster ovary cell.

10. serum-free animal cell culture medium dry powder according to claim 6 wherein, is benchmark with the volume of said substratum solvent, and said serum-free animal cell culture medium dry powder comprises the component shown in the following table 4:

Table 4

Wherein, the said serum-free animal cell culture medium dry powder culture medium dry powder that is the BHK21 cell.

11. the preparation method of a serum-free animal cell culture medium dry powder, this method comprise the component that is selected from any described serum-free animal cell culture medium dry powder among the claim 1-10 is dispersed in the dispersion agent, removes the dispersion agent in the gained mixture.

12. method according to claim 11 is characterized in that, one or more in alcohol, alkaline aqueous solution and the acidic aqueous solution that said dispersion agent is selected from zero(ppm) water, deionized water, water for injection, redistilled water, ultrapure water, can dissolve each other with any ratio with water.

13. method according to claim 12; Wherein, Said alkaline aqueous solution is the aqueous solution of the cationic alkali that occurs in the serum-free animal cell culture medium dry powder described in sodium hydroxide solution or the claim 1-10, and said acidic aqueous solution is the aqueous acid of the acid ion that occurs of the serum-free animal cell culture medium dry powder described in the claim 1-10.

14. liquid serum-free animal cell culture medium; Said liquid serum-free animal cell culture medium comprises the substratum solvent and is dispersed in the serum-free animal cell culture medium dry powder in this substratum solvent; Wherein, Said substratum solvent is selected from one or more in zero(ppm) water, deionized water, water for injection, redistilled water and the ultrapure water; It is characterized in that said serum-free animal cell culture medium dry powder is one or more in any described serum-free animal cell culture medium dry powder among the claim 1-10.

15. the preparation method of a liquid serum-free animal cell culture medium; This method comprises serum-free animal cell culture medium dry powder is dispersed in the substratum solvent; Filtration sterilization, wherein, said substratum solvent is selected from one or more in zero(ppm) water, deionized water, water for injection, redistilled water and the ultrapure water; It is characterized in that said serum-free animal cell culture medium dry powder is selected from one or more in the serum-free animal cell culture medium dry powder described in the claim 1-10.