CN101386836B - Zooblast culture medium dry powder composition, culture medium composition and preparation method thereof - Google Patents

Zooblast culture medium dry powder composition, culture medium composition and preparation method thereof Download PDF

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CN101386836B
CN101386836B CN2007101217097A CN200710121709A CN101386836B CN 101386836 B CN101386836 B CN 101386836B CN 2007101217097 A CN2007101217097 A CN 2007101217097A CN 200710121709 A CN200710121709 A CN 200710121709A CN 101386836 B CN101386836 B CN 101386836B
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culture medium
dry powder
powder composition
zooblast
zooblast culture
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CN101386836A (en
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张韧
陈文庆
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Merck Millipore Beijing Skywing Co ltd
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Beijing Skywing Technology Co Ltd
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Abstract

The invention provides a dry powder composition for a zooblast culture medium. Compared with the prior dry powder composition for the zooblast culture medium, the dry powder composition provided by the invention reduces the serum dosage of animals by introduction of recombinant protein or animal origin compositions, the dry powder composition for the zooblast culture medium can be matched with low-content animal serum for use without additional introduction of protein compositions (such as the recombinant protein, plant protein or the animal origin compositions including cytokines and so on), and has the same or better function on promoting the growth of zooblast compared with high-content animal serum, so that the dry powder composition for the zooblast culture medium does not generate side effects along with the protein compositions, and has good safety and low cost. Cells cultured by the dry powder composition for the zooblast culture medium have small difference for different batches, low cost and good safety, are favorable for separating downstream products of the cells, and are suitable to be used for virus hosts, expression vectors and so on of biological products and other biological products.

Description

Zooblast culture medium dry powder composition, culture media composition and preparation method thereof
Technical field
The present invention relates to cell culture medium dry powder composite, cell cultures based composition and use thereof in packaging and their preparation method, relate in particular to Zooblast culture medium dry powder composition, Zooblast culture medium composition and their preparation method.
Background technology
In the existing animal cell culture, generally Zooblast culture medium dry powder composition is scattered in formation liquid culture based composition and use thereof in packaging in the supporting agent (as redistilled water), the animal serum with final concentration at least 10 volume % is used then; Perhaps will not contain the zooblast liquid culture based composition and use thereof in packaging of animal serum and the animal serum of final concentration at least 10 volume % is used; Perhaps directly use the liquid culture based composition and use thereof in packaging of the animal serum that comprises final concentration at least 10 volume %, could guarantee that the normal growth of cell goes down to posterity.
Yet animal serum indefinite complex mixture that is composition.Animal serum for example contains on the one hand, carries the translocator of hormone, mineral substance, trace element, lipid material; The cell attachment factor and spreading factor etc. can promote cell growth, stable detoxifcation, keep the pH value of substratum, and the direct or indirect enzymolysis of arrestin enzyme.But then, for example there are a lot of problems in animal serum, adding to reach causes the medium pH value to change greatly in the process of replenishing, be subject to other microbial contaminations in the storage process, after thawing, low tempertaure storage easily produces precipitation, different batches difference is big, the cost height, and be difficult to removal, poor stability etc. from the cell derived product.
Therefore there is the trend that as far as possible uses no or little animal serum in the Zooblast culture medium.Prior art is generally by extra recombinant protein and the animal derived components such as Regular Insulin, Recombulin, people source Transferrins,iron complexes, bovine serum albumin, cytokine, somatomedin of adding, reduce the amount of using animal serum, a kind of culture media composition that is derived from the progenitor cell/stem cell of muscle tissue is for example disclosed among the CN 1723277, add Regular Insulin, FGF type somatomedin in the said composition, can reduce the usage quantity of human serum.But recombinant protein and animal derived components have a significant impact the security of biological products (such as vaccine), cause allergic reaction etc. such as recombinant protein and animal derived components.
To sum up, the zooblast liquid culture based composition and use thereof in packaging that needs to cooperate the Zooblast culture medium dry powder composition of low levels animal serum (final concentration is less than 10 volume %) use or do not contain animal serum, perhaps the zooblast liquid culture based composition and use thereof in packaging of low levels animal serum (final concentration is less than 10 volume %).
Summary of the invention
The objective of the invention is to overcome existing Zooblast culture medium dry powder composition and must be used the shortcoming that could promote zooblast growth, poor stability with the high-content animal serum, a kind of Zooblast culture medium dry powder composition is provided, and said composition cooperates the animal serum concentration of low levels just can promote that zooblast is grown, security is good.
Second purpose of the present invention provides the preparation method of above-mentioned Zooblast culture medium dry powder composition.
The 3rd purpose of the present invention provides the Zooblast culture medium composition that comprises above-mentioned Zooblast culture medium dry powder composition.
The 4th purpose of the present invention provides the Zooblast culture medium preparation of compositions that comprises above-mentioned Zooblast culture medium dry powder composition method.
The present inventor is surprised to find that, only Zooblast culture medium dry powder composition is adjusted according to the component and the content range of table 1, add supporting agent and be used, just can reach the Zooblast culture medium and the low levels animal serum that contain recombinant protein and animal derived components and be used the same promotion cell growth effect that (final concentration 1 volume % is extremely less than 10 volume %) or existing Zooblast culture medium and high-content animal serum (final concentration at least 10 volume %) are used with low levels animal serum (final concentration 1 to less than 10 volume %).
The invention provides a kind of Zooblast culture medium dry powder composition, wherein, this Zooblast culture medium dry powder composition comprises the component shown in the following table 1:
Table 1
Component Weight part Component Weight part
The L-L-Ala 80-120 Calcium nitrate tetrahydrate 30-80
The L-arginine monohydrochloride 180-210 Repone K 320-500
The L-aspartic acid 5-30 Anhydrous magnesium sulfate 40-80
The L-asparagine 30-60 Sodium-chlor 6000-8000
The L-cysteine hydrochloride 10-30 Disodium hydrogen phosphate,anhydrous 300-400
L-L-glutamic acid 100-150 Iron vitriol 0.1-1
L-glutaminate 200-500 Zinc vitriol 0.2-3
The L-L-Histidine hydrochloride 20-50 D-glucose 1000-3000
The L-oxyproline 2-15 Gsh 0.2-2
The L-Isoleucine 90-150 Sodium.alpha.-ketopropionate 20-120
The L-leucine 10-70 Xitix 1-10
L lysine HCL 100-200 Choline chloride 60 5-25
The L-methionine(Met) 10-50 The D-calcium pantothenate 1-10
The L-phenylalanine 10-50 Folic acid 2-10
The L-proline(Pro) 20-60 Niacinamide 1-5
The L-Serine 20-60 Pyridoxine hydrochloride 1-10
The L-Threonine 60-100 Riboflavin 0.2-2
Component Weight part Component Weight part
The L-tryptophane 10-50 Thiamine hydrochloride 1-10
The L-Xie Ansuan 30-70 Vitamin B12 1-10
L-tyrosine 30-80 Magnesium dichloride hexahydrate 40-100
Glycine 30-80 Xanthoglobulin 1-10
The L-cystine hydrochloride 30-80 Thymidine 0.1-2
Calcium Chloride Powder Anhydrous 30-80 Anhydrous potassium dihydrogenphosphate 20-100
AMSP 30-80
The present invention also provides the preparation method of above-mentioned Zooblast culture medium dry powder composition, and this method comprises that the component with Zooblast culture medium dry powder composition 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 Zooblast culture medium composition, said composition comprises Zooblast culture medium dry powder composition and supporting agent thereof, wherein, described Zooblast culture medium dry powder composition is selected from one or more in the Zooblast culture medium dry powder composition of the present invention.
The present invention also provides above-mentioned Zooblast culture medium preparation of compositions method, this method comprises Zooblast culture medium dry powder composition is dispersed in the supporting agent, filtration sterilization, wherein, described Zooblast culture medium dry powder composition is selected from one or more in the Zooblast culture medium dry powder composition of the present invention.
Compare by introducing the Zooblast culture medium dry powder composition that recombinant protein or animal-origin component reduce the animal serum consumption with existing, because Zooblast culture medium dry powder composition of the present invention, need not additionally to introduce protein ingredient (recombinant protein for example, vegetable-protein or animal-origin component such as cytokine etc.), can cooperate the low levels animal serum to use to reach the effect that maintains an equal level with the high-content animal serum even better promote the zooblast growth, therefore Zooblast culture medium dry powder composition of the present invention does not produce the side effect of following protein ingredient, and security is good and cost is low.With Zooblast culture medium dry powder composition cultured cells of the present invention, different batches difference is little, and cost is low, is beneficial to the isolated cell derived product, security is good, is suitable for use as the virus host of producing biological products such as vaccine, expression vector etc.
Description of drawings
Fig. 1 is the Zooblast culture medium dry powder composition of the use embodiment of the invention 1, also the photo of 72 hours Vero cell of animal serum concentration (3 volume % calf serum) cultivation is hanged down in cooperation;
Fig. 2 is Zooblast culture medium dry powder composition that uses Comparative Examples 1 and the photo that cooperates 72 hours Vero cell of high animal serum concentration (10 volume % calf serum) cultivation;
Fig. 3 cultivates the photo of 72 hours Vero cell for using CN 1723277 culture media compositions (containing cytokine+hormone+low-serum-concentration 3 volume % calf serums);
Fig. 4 is the Zooblast culture medium dry powder composition of use Comparative Examples 1, also the photo of 72 hours Vero cell of animal serum concentration (3 volume % calf serum) cultivation is hanged down in cooperation;
Fig. 5 cultivates the photo of 72 hours Chinese hamster ovary celI for the Zooblast culture medium composition (containing 2.9 volume % calf serums) that uses embodiment 5.
Embodiment
The invention provides a kind of Zooblast culture medium dry powder composition, wherein, this Zooblast culture medium dry powder composition comprises the component shown in the following table 1:
Table 1
Component Weight part Component Weight part
The L-L-Ala 80-120 Calcium nitrate tetrahydrate 30-80
The L-arginine monohydrochloride 180-210 Repone K 320-500
The L-aspartic acid 5-30 Anhydrous magnesium sulfate 40-80
The L-asparagine 30-60 Sodium-chlor 6000-8000
The L-cysteine hydrochloride 10-30 Disodium hydrogen phosphate,anhydrous 300-400
L-L-glutamic acid 100-150 Iron vitriol 0.1-1
L-glutaminate 200-500 Zinc vitriol 0.2-3
The L-L-Histidine hydrochloride 20-50 D-glucose 1000-3000
The L-oxyproline 2-15 Gsh 0.2-2
The L-Isoleucine 90-150 Sodium.alpha.-ketopropionate 20-120
The L-leucine 10-70 Xitix 1-10
L lysine HCL 100-200 Choline chloride 60 5-25
The L-methionine(Met) 10-50 The D-calcium pantothenate 1-10
The L-phenylalanine 10-50 Folic acid 2-10
The L-proline(Pro) 20-60 Niacinamide 1-5
The L-Serine 20-60 Pyridoxine hydrochloride 1-10
The L-Threonine 60-100 Riboflavin 0.2-2
Component Weight part Component Weight part
The L-tryptophane 10-50 Thiamine hydrochloride 1-10
The L-Xie Ansuan 30-70 Vitamin B12 1-10
L-tyrosine 30-80 Magnesium dichloride hexahydrate 40-100
Glycine 30-80 Xanthoglobulin 1-10
The L-cystine hydrochloride 30-80 Thymidine 0.1-2
Calcium Chloride Powder Anhydrous 30-80 Anhydrous potassium dihydrogenphosphate 20-100
AMSP 30-80
Preferred described cell culture medium dry powder composite comprises the component shown in the following table 2:
Table 2
Component Weight part Component Weight part
The L-L-Ala 90-110 Calcium nitrate tetrahydrate 30-60
The L-arginine monohydrochloride 190-210 Repone K 320-400
The L-aspartic acid 5-20 Anhydrous magnesium sulfate 50-70
The L-asparagine 30-50 Sodium-chlor 6500-7500
The L-cysteine hydrochloride 10-15 Disodium hydrogen phosphate,anhydrous 300-380
L-L-glutamic acid 100-120 Iron vitriol 0.1-0.5
L-glutaminate 300-500 Zinc vitriol 0.2-1.5
The L-L-Histidine hydrochloride 30-50 D-glucose 2000-3000
The L-oxyproline 2-10 Gsh 0.6-1.5
The L-Isoleucine 90-120 Sodium.alpha.-ketopropionate 50-100
The L-leucine 30-70 Xitix 2-7
L lysine HCL 100-180 Choline chloride 60 10-16
Component Weight part Component Weight part
The L-methionine(Met) 20-50 The D-calcium pantothenate 2-6
The L-phenylalanine 30-50 Folic acid 3-6
The L-proline(Pro) 40-60 Niacinamide 2-4
The L-Serine 40-60 Pyridoxine hydrochloride 2-6
The L-Threonine 60-80 Riboflavin 0.5-1
The L-tryptophane 10-30 Thiamine hydrochloride 2-5
The L-Xie Ansuan 40-70 Vitamin B12 3-7
L-tyrosine 30-50 Magnesium dichloride hexahydrate 50-80
Glycine 30-60 Xanthoglobulin 1-4
The L-cystine hydrochloride 40-60 Thymidine 0.5-1
Calcium Chloride Powder Anhydrous 60-80 Anhydrous potassium dihydrogenphosphate 50-90
AMSP 40-70
Preferred described cell dry powder composite can also comprise one or more in the vitamin K3 of vitamin D2,1-3 weight part of linolic acid, the 1-3 weight part of 1-3 weight part and 2-10 weight part phenol red.Wherein, linolic acid, vitamin D2 and vitamin K3 can further promote the growth of cell; Phenol red as the adding of pH pH indicator, can be by estimating the growing state that grasp cell at any time.More preferably described cell dry powder composite can also comprise one or more in the vitamin K3 of vitamin D2,1.5-2 weight part of linolic acid, the 1.5-2 weight part of 1.5-2 weight part and 3-5 weight part phenol red.
The preparation Zooblast culture medium dry powder composition 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 milling to mix) of blended solid component under the exsiccant condition.When using dry method, preferred at first dry respectively to them according to the character of each component that constitutes culture medium dry powder composition.Such as can be at 100-120 ℃ with AMSP, Repone K, anhydrous magnesium sulfate, sodium-chlor, disodium hydrogen phosphate,anhydrous, 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, L-proline(Pro), L-Serine, L-tryptophane, L-Threonine, L-Xie Ansuan, L-tyrosine, glycine, L-cystine hydrochloride were descended dry 1-3 hour at 30-80 ℃ of preferred 35-50 ℃; Anhydrous potassium dihydrogenphosphate is put into vacuum drying oven with calcium nitrate tetrahydrate, gsh, iron vitriol, Zinc vitriol, Sodium.alpha.-ketopropionate and is vacuumized dry more than 10 hours.So-called wet method refers to form the solid ingredient and the dispersant of Zooblast culture medium dry powder composition, removes the dispersion agent of gained mixture then.Dry method can also be combined with wet method, be about to a part of solid ingredient wet-mixed, remove dispersion agent after, mix with the solid ingredient of rest part with dry method.
The present invention also provides a kind of preferred preparation method of Zooblast culture medium dry powder composition, this method comprises that this method comprises that the component with Zooblast culture medium dry powder composition of the present invention is dispersed in the dispersion agent, removes the dispersion agent in the gained mixture.The dispersion of the component of described Zooblast culture medium dry powder composition in dispersion agent can realize by this area various dispersing method commonly used, such as stirring, sonic oscillation etc.
Described dispersion agent can be selected from the various dispersion agents that this area is used to prepare Zooblast culture medium dry powder composition.The effect of described dispersion agent is that the various components of Zooblast culture medium dry powder composition are mixed equably, generally dispersion agent there is not special requirement, only otherwise introduce the new ion that influences the cell growth, pollute impurity (as intracellular toxin, microorganism etc.), do not change pH value scope that this Zooblast culture medium dry powder composition is dissolved in the suitable cell growth behind the supporting agent, removal gets final product easily under the prerequisite of not destroying nutrient media components.Therefore described dispersion agent be preferably selected from distilled 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 Zooblast culture medium dry powder composition of the present invention, the aqueous acid of the acid ion that described acidic aqueous solution occurs for the Zooblast culture medium dry powder composition described in the present invention.The described alcohol that can dissolve each other with any ratio with water can be one or more in ethanol, methyl alcohol, the propyl alcohol.In the aqueous solution of the aqueous solution of the aqueous solution of the preferred sodium hydroxide of described alkaline aqueous solution, the aqueous solution of potassium 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 described 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 that constitutes Zooblast culture medium dry powder composition adds in the dispersion agent.In addition, the various components that constitute Zooblast culture medium dry powder composition can be dispersed in in a kind of dispersion agent, also different components can be dispersed in the different dispersion agents, mix the dispersion that obtains then.The preferred latter for example preferably is scattered in 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 ℃; Lipophilic substance (as vitamin K3, vitamin D2) is scattered in earlier in the hydrophilic small molecules organic solvent of lower boiling (as ethanol).For the component (being no more than the component of 10 weight parts such as content) seldom of content in the described culture media composition, in the preparation, can prepare the high concentration liquid dispersion of this component earlier with dispersion agent, according to the required quantitative form of this component in the final culture medium dry powder composition this component be mixed with other components then with this high concentration liquid dispersion.The temperature of used dispersion agent should not be higher than by the lowest decomposition temperature of dispersed component.
The consumption of described dispersion agent has no particular limits, as long as can make the component of culture medium dry powder composition and the dispersion that dispersion agent forms stable homogeneous; In addition, satisfying under the prerequisite of aforementioned condition, for 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.Described drying can be heating, vacuum-drying, vacuum lyophilization, siccative (as silica gel, alumina gel, molecular sieve, gac, bone black, charcoal, atlapulgite, five phosphorus oxide, unslaked lime, calcium chloride etc.) moisture absorption etc.
The present invention also provides a kind of Zooblast culture medium composition, said composition comprises Zooblast culture medium dry powder composition and supporting agent thereof, wherein, one or more in the described Zooblast culture medium dry powder composition Zooblast culture medium dry powder composition of the present invention.
Described supporting agent can be selected from the various supporting agents of this area as culturing cell, is preferably selected from distilled water, deionized water, water for injection, redistilled water and the ultrapure water one or more, and more preferably described supporting agent is water for injection and/or ultrapure water.The consumption of described supporting agent is the 600000-1500000 weight part, is preferably the 800000-1200000 weight part, more preferably 1000000 weight parts.Unit weight part that each component adopted in unit weight part of described supporting agent and table 1 and the table 2 is identical.
Described Zooblast culture medium composition can also comprise that final concentration is 1 volume % to the animal serum less than 10 volume %, and preferably including final concentration is the animal serum of 3 volume % to 5 volume %.Described animal serum can be one or more in bovine serum (as foetal calf serum), horse serum, rabbit anteserum, monkey serum and the people source serum.
Zooblast culture medium composition of the present invention can be with the component of Zooblast culture medium dry powder composition of the present invention, and weighing one by one is dispersed in the identical supporting agent then; The component of described Zooblast culture medium dry powder composition is dispersed in respectively in the different supporting agents, mixes different dispersions then.For the component (being no more than the component of 10 weight parts such as content) seldom of content in the described culture media composition, in the preparation, can prepare the high concentration liquid dispersion of this component earlier with supporting agent, then according to component in the final liquid nutrient medium required quantitatively add this component with the form of this high concentration liquid dispersion.The temperature of used supporting agent should not be higher than by the lowest decomposition temperature of dispersed component.Zooblast culture medium preparation of compositions method more preferably provided by the invention, this method comprises Zooblast culture medium dry powder composition is dispersed in the supporting agent, filtration sterilization, wherein, described Zooblast culture medium dry powder composition is selected from one or more in the Zooblast culture medium dry powder composition of the present invention.Preferred this method also comprises, before filtration sterilization, in the supporting agent that is dispersed with described culture medium dry powder composition, add final concentration and be 1 volume % to animal serum, more preferably add and comprise that final concentration is the animal serum of 3 weight % to 5 weight % less than 10 volume %.The method of described 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 described Zooblast culture medium dry powder composition in supporting agent can realize by this area various dispersing method commonly used, such as stirring, sonic oscillation etc.
Zooblast culture medium dry powder composition of the present invention, Zooblast culture medium composition (liquid) are to can cultured cells having no particular limits, can cultivate somatocyte (as the Vero cell) or sexual cell (Chinese hamster ovary celI), primary cell (as 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 (as the BHK21 cell) of process artificially breeding.
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, purity requirement height not only, preferred analytical pure, and can not have any material that influences the cell normal physiological activity, therefore also need to 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 illustrate Zooblast culture medium dry powder composition of the present invention and preparation method thereof.
Table 3
Component mg/L Component mg/L
The L-L-Ala 90 Calcium nitrate tetrahydrate 50
The L-arginine monohydrochloride 200 Repone K 400
The L-aspartic acid 20 Anhydrous magnesium sulfate 43
The L-asparagine 30 Sodium-chlor 6400
The L-cysteine hydrochloride 20 Disodium hydrogen phosphate,anhydrous 320
Component mg/L Component mg/L
L-L-glutamic acid 130 Iron vitriol 0.2
L-glutaminate 300 Zinc vitriol 0.3
The L-L-Histidine hydrochloride 30 D-glucose 2000
The L-oxyproline 10 Gsh 0.3
The L-Isoleucine 120 Sodium.alpha.-ketopropionate 50
The L-leucine 30 Xitix 3
L lysine HCL 150 Choline chloride 60 15
The L-methionine(Met) 20 The D-calcium pantothenate 3
The L-phenylalanine 30 Folic acid 5
The L-proline(Pro) 40 Niacinamide 5
The L-Serine 30 Pyridoxine hydrochloride 3
The L-Threonine 80 Riboflavin 0.6
The L-tryptophane 20 Thiamine hydrochloride 2
The L-Xie Ansuan 50 Vitamin B12 2
L-tyrosine 32 Magnesium dichloride hexahydrate 60
Glycine 50 Xanthoglobulin 2
The L-cystine hydrochloride 50 Thymidine 1
Calcium Chloride Powder Anhydrous 60 Anhydrous potassium dihydrogenphosphate 25
AMSP 40
The mg/L of unit of solid ingredient is meant in table 3 and following each table, with the volume with this culture medium dry powder composition and the final liquid nutrient medium of supporting agent mixing gained is benchmark, with respect to every liter of final liquid nutrient medium, the milligram number of the solid matter that when the preparation substratum, need add.With the consumption of the solid ingredient shown in the table 3 according to 10 liters of final liquid nutrient mediums, after accurately weighing mixes one by one with ten thousand/balance, add 10 liters of distilled water again, after stirring 2 hours with Mei Ying Pu, the Shanghai 85-2 of instrument Manufacturing Co., Ltd type agitator, 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 Zooblast culture medium dry powder composition of the present invention.
Embodiment 2
Present embodiment is used to illustrate Zooblast culture medium dry powder composition of the present invention and preparation method thereof.
Table 4
Component mg/L Component mg/L
The L-L-Ala 110 Calcium nitrate tetrahydrate 60
The L-arginine monohydrochloride 190 Repone K 320
The L-aspartic acid 10 Anhydrous magnesium sulfate 60
The L-asparagine 50 Sodium-chlor 7300
The L-cysteine hydrochloride 10 Disodium hydrogen phosphate,anhydrous 380
L-L-glutamic acid 100 Iron vitriol 0.4
L-glutaminate 500 Zinc vitriol 0.8
The L-L-Histidine hydrochloride 45 D-glucose 3000
The L-oxyproline 5 Gsh 0.9
The L-Isoleucine 90 Sodium.alpha.-ketopropionate 60
The L-leucine 70 Xitix 7
L lysine HCL 100 Choline chloride 60 10
The L-methionine(Met) 40 The D-calcium pantothenate 4
The L-phenylalanine 50 Folic acid 3
The L-proline(Pro) 60 Niacinamide 4
The L-Serine 60 Pyridoxine hydrochloride 5
The L-Threonine 60 Riboflavin 1
The L-tryptophane 15 Thiamine hydrochloride 5
The L-Xie Ansuan 70 Vitamin B12 5
Component mg/L Component mg/L
L-tyrosine 50 Magnesium dichloride hexahydrate 80
Glycine 30 Xanthoglobulin 4
The L-cystine hydrochloride 60 Thymidine 0.5
Calcium Chloride Powder Anhydrous 80 Anhydrous potassium dihydrogenphosphate 60
AMSP 60 Phenol red 5
Linolic acid 3 Vitamin D2 1
Solid ingredient shown in the table 4 takes by weighing with ten thousand/analytical balance according to the consumption of 10 liters of final liquid nutrient mediums.Wherein, 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, the L-proline(Pro), the L-oxyproline, the L-Serine, the L-tryptophane, the L-Threonine, the L-Xie Ansuan, xitix, choline chloride 60, the D-calcium pantothenate, folic acid, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12 is dissolved in and obtains B solution in the 3000ml distilled water; The ethanol that vitamin D2, linolic acid is dissolved in 10ml 99% obtains C solution; Merge A solution, B solution, C solution, in the gained mixing solutions, add glucose then, AMSP, Repone K, anhydrous magnesium sulfate, Calcium Chloride Powder Anhydrous, sodium-chlor, disodium hydrogen phosphate,anhydrous, anhydrous potassium dihydrogenphosphate, glycine, the L-cystine hydrochloride, the L-cysteine hydrochloride, L-tyrosine, the L-asparagine, calcium nitrate tetrahydrate, gsh, iron vitriol, Zinc vitriol, Sodium.alpha.-ketopropionate, phenol red, being settled to 10000ml with distilled 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 Zooblast culture medium dry powder composition of the present invention.
Embodiment 3
Present embodiment is used to illustrate Zooblast culture medium dry powder composition of the present invention and preparation method thereof.
Table 5
Component mg/L Component mg/L
The L-L-Ala 100 Calcium nitrate tetrahydrate 40
The L-arginine monohydrochloride 205 Repone K 350
The L-aspartic acid 15 Anhydrous magnesium sulfate 55
The L-asparagine 40 Sodium-chlor 7000
The L-cysteine hydrochloride 15 Disodium hydrogen phosphate,anhydrous 350
Component mg/L Component mg/L
L-L-glutamic acid 120 Iron vitriol 0.3
L-glutaminate 400 Zinc vitriol 0.5
The L-L-Histidine hydrochloride 35 D-glucose 2500
The L-oxyproline 8 Gsh 0.6
The L-Isoleucine 100 Sodium.alpha.-ketopropionate 80
The L-leucine 50 Xitix 5
L lysine HCL 130 Choline chloride 60 13
The L-methionine(Met) 30 The D-calcium pantothenate 5
The L-phenylalanine 40 Folic acid 4
The L-proline(Pro) 50 Niacinamide 3
The L-Serine 50 Pyridoxine hydrochloride 4
The L-Threonine 70 Riboflavin 0.8
The L-tryptophane 28 Thiamine hydrochloride 4
The L-Xie Ansuan 60 Vitamin B12 6
L-tyrosine 40 Magnesium dichloride hexahydrate 70
Glycine 40 Xanthoglobulin 3
The L-cystine hydrochloride 55 Thymidine 0.8
Calcium Chloride Powder Anhydrous 70 Anhydrous potassium dihydrogenphosphate 50
AMSP 50 Phenol red 4
Linolic acid 2 Vitamin D2 2
Vitamin K3 2
Solid ingredient shown in the table 5 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 xanthoglobulin, thymidine; Vitamin K3, vitamin D2, linolic acid are dissolved in 10ml 99% ethanolic soln, obtain B solution; With xitix, choline chloride 60, the D-calcium pantothenate, folic acid, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12 is dissolved in and obtains C solution in the 1000ml distilled water, merge A solution, B solution and C solution, in the gained mixing solutions, add glucose then, being settled to 10000ml with distilled 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-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, disodium hydrogen phosphate,anhydrous, anhydrous potassium dihydrogenphosphate, Calcium Chloride Powder Anhydrous, in 105 ℃ of dryings 2 hours, with the L-L-Ala, the L-aspartic acid, L-L-glutamic acid, L-glutaminate, the L-L-Histidine hydrochloride, the L-arginine monohydrochloride, the L-Isoleucine, the L-leucine, L lysine HCL, the L-methionine(Met), the L-phenylalanine, the L-proline(Pro), the L-Serine, the L-tryptophane, the L-Threonine, the L-Xie Ansuan, L-tyrosine, glycine, the L-cystine hydrochloride, phenol red following dry 1 hour in 40 ℃.Calcium nitrate tetrahydrate, Magnesium dichloride hexahydrate, gsh, iron vitriol, Zinc vitriol, Sodium.alpha.-ketopropionate, L-cysteine hydrochloride, L-asparagine, L-oxyproline 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 Zooblast culture medium dry powder composition of the present invention.
Comparative Examples 1
This Comparative Examples is used to illustrate existing Zooblast culture medium dry powder composition (commodity are called MD500 type 199 substratum) and preparation method thereof.
Table 6
Component (mg/L) Component (mg/L)
Calcium chloride 200.00 D-glucose 1000.00
Nine nitric hydrate iron 0.70 Gsh (reduced form) 0.05
Repone K 400.00 Guanine hydrochloride 0.30
Anhydrous magnesium sulfate 97.67 Xanthoglobulin 0.30
Sodium-chlor 6800.00 Phenol red 20.00
AMSP 121.74 D-ribose 0.50
The L-L-Ala 25.00 Sodium acetate 50.00
The L-arginine monohydrochloride 70.00 Thymus pyrimidine 0.30
The L-aspartic acid 30.00 Tween 80 20.00
The L-cysteine hydrochloride 0.11 Uridylic 0.30
The L-cystine hydrochloride 26.00 Xanthine 0.30
Component (mg/L) Component (mg/L)
L-L-glutamic acid 75.00 Vitamins C 0.05
L-glutaminate 100.00 Vitamin-E 0.01
Glycine 50.00 Vitamin H 0.01
The L-L-Histidine hydrochloride 21.88 Vitamin D2 0.10
The L-oxyproline 10.00 The D-calcium pantothenate 0.01
The L-Isoleucine 40.00 Choline chloride 60 0.50
The L-leucine 60.00 Folic acid 0.01
L lysine HCL 70.00 The i-inositol 0.05
The L-methionine(Met) 15.00 Vitamin K3 0.01
The L-phenylalanine 25.00 Nicotinic acid 0.025
The L-proline(Pro) 40.00 Niacinamide 0.025
The L-Serine 25.00 Para-amino benzoic acid 0.05
The L-Threonine 30.00 Pyridoxal hydrochloride 0.025
The L-tryptophane 10.00 Pyridoxine hydrochloride 0.025
L-tyrosine 40.00 Riboflavin 0.01
The L-Xie Ansuan 25.00 Thiamine hydrochloride 0.01
Adenine sulfate 10.00 Vitamin(e) A acetate 0.14
Adenylic acid (AMP) 0.20 Cholesterol 0.20
Sodium ATP 1.00 Ribodesose 0.50
Table 6 is the component of trade(brand)name 199 substratum, and the consumption according to 10 liters of final liquid nutrient mediums takes by weighing with ten thousand/analytical balance.Cholesterol,, vitamin-E, vitamin D2, vitamin K3 and tween 80 are dissolved in 0.1 liter of ethanol; Thymus pyrimidine, uridylic, xanthine and xanthoglobulin adding are contained in 1 liter of redistilled water of 0.5 milliliter of ammoniacal liquor, and 60 ℃ are heated to dissolving; Make the vibration of folic acid and riboflavin be dissolved in 2.5 liters of redistilled waters; Vitamin H and guanine hydrochloride are joined in the hydrochloric acid soln of 2 liters of 0.075N, and 50 ℃ are heated to dissolving; Sodium ATP, adenylic acid (AMP), ribodesose, gsh, D-ribose, D-calcium pantothenate, vitamins C, folic acid, choline chloride 60, i-inositol, nicotinic acid, niacinamide, para-amino benzoic acid, pyridoxal hydrochloride, pyridoxine hydrochloride and thiamine hydrochloride are dissolved in 200 milliliters of redistilled waters; Merge five kinds of solution that obtain, add D-glucose then, be settled to 10 liters with distilled water and shake up.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 ℃, slowly heating up kept under the vacuum tightness 8Pa lyophilize 24 hours, obtained lyophilized powder.
With sodium-chlor, Repone K, anhydrous magnesium sulfate, adenine sulfate, nine nitric hydrate iron, calcium chloride, sodium acetate, AMSP, in 105 ℃ of baking ovens dry 2 hours.With the L-L-Ala, L-glutaminate, the L-Isoleucine, the L-methionine(Met), the L-oxyproline, the L-proline(Pro), the L-Serine, the L-tryptophane, the L-Xie Ansuan, the L-arginine monohydrochloride, the L-cysteine hydrochloride, the L-cystine hydrochloride, L-L-glutamic acid, the L-L-Histidine hydrochloride, L lysine HCL, the L-aspartic acid, glycine, the L-leucine, the L-phenylalanine, the L-proline(Pro), the L-Threonine, L-tyrosine, phenol redly vacuumized dry 15 hours with vacuum drying oven.
Above-mentioned obtained freeze-drying powder and drying solid component are carried out mixing in continuous 10 hours in ball mill, and the gained pressed powder is existing Zooblast culture medium dry powder composition, trade(brand)name MD500 type 199 substratum.
Embodiment 4
Present embodiment is used to illustrate Zooblast culture medium composition and method of making the same of the present invention.
Dry powder composite 123.64 grams with embodiment 3 makes are dissolved in 9 liters of distilled water, are settled to 10 liters of mixings with distilled water then, use the bacteriological filtration membrane filtration sterilization of 0.22 μ m, promptly obtain Zooblast culture medium composition of the present invention.
Embodiment 5
Present embodiment is used to illustrate Zooblast culture medium composition and method of making the same of the present invention.
Table 7
Component mg/L Component mg/L
The L-L-Ala 80 Calcium nitrate tetrahydrate 30
The L-arginine monohydrochloride 210 Repone K 380
The L-aspartic acid 7 Anhydrous magnesium sulfate 70
The L-asparagine 45 Sodium-chlor 6500
The L-cysteine hydrochloride 13 Disodium hydrogen phosphate,anhydrous 330
L-L-glutamic acid 110 Iron vitriol 0.5
L-glutaminate 450 Zinc vitriol 1.2
The L-L-Histidine hydrochloride 50 D-glucose 2700
The L-oxyproline 3 Gsh 1.2
The L-Isoleucine 110 Sodium.alpha.-ketopropionate 100
Component mg/L Component mg/L
The L-leucine 60 Xitix 6
L lysine HCL 180 Choline chloride 60 12
The L-methionine(Met) 50 The D-calcium pantothenate 6
The L-phenylalanine 45 Folic acid 6
The L-proline(Pro) 55 Niacinamide 2
The L-Serine 40 Pyridoxine hydrochloride 6
The L-Threonine 75 Riboflavin 0.5
The L-tryptophane 30 Thiamine hydrochloride 3
The L-Xie Ansuan 40 Vitamin B12 4
L-tyrosine 30 Magnesium dichloride hexahydrate 50
Glycine 60 Xanthoglobulin 1
The L-cystine hydrochloride 40 Thymidine 0.6
Calcium Chloride Powder Anhydrous 75 Anhydrous potassium dihydrogenphosphate 70
AMSP 70 Phenol red 6
Linolic acid 1.5 Vitamin D2 3
Vitamin K3 3
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, 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, the L-proline(Pro), the L-oxyproline, the L-Serine, the L-tryptophane, the L-Threonine, the L-Xie Ansuan, xitix, choline chloride 60, the D-calcium pantothenate, folic acid, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin B12 is dissolved in and obtains B solution in the 3000ml distilled water; The ethanol that vitamin D2, linolic acid, vitamin K3 is dissolved in 10ml 99% obtains C solution; Merge A solution, B solution, C solution, in the gained mixing solutions, add glucose, AMSP, Repone K, anhydrous magnesium sulfate, Calcium Chloride Powder Anhydrous, sodium-chlor, disodium hydrogen phosphate,anhydrous, anhydrous potassium dihydrogenphosphate, glycine, L-cystine hydrochloride, L-cysteine hydrochloride, L-tyrosine, L-asparagine, calcium nitrate tetrahydrate, gsh, iron vitriol, Zinc vitriol, Sodium.alpha.-ketopropionate, phenol red then, be settled to 10000ml with distilled water and shake up.The 300ml calf serum is added gained substratum mixing, use the bacteriological filtration membrane filtration of 0.22 μ m to sterilize in the gained mixture, obtain the culture media composition of the present invention that 10300ml contains 2.9% bovine serum.
Performance test:
(1) clarity
According to two appendix IX of Pharmacopoeia of People's Republic of China version in 2005 B clarity test procedure, detect the culture medium dry powder composition of the Comparative Examples 1 of new system and embodiment 1-3 and the culture medium dry powder composition of Comparative Examples 1 after storing 30 days under the environment of room temperature, relative humidity 50% and embodiment 1-3 respectively and mixes the medium liquid that the back forms with distilled water, and the culture media composition (liquid) of the embodiment 4-5 after one week of preservation under 2 ℃ behind the culture media composition (liquid) of new system embodiment 4-5 and the filtration sterilization.For culture medium dry powder composition, get the consumption of 1 liter of preparation, place the 1000ml beaker, add water 1000ml, be stirred to dissolving; Directly test for culture media composition (liquid).Test result is as shown in the table:
Table 8
Sample Embodiment 1 Storage implementation example 1 Embodiment 2 Storage implementation example 2 Embodiment 3 Storage implementation example 3 Comparative Examples 1 Storage Comparative Examples 1 Embodiment 4 Storage implementation example 4 Embodiment 5 Storage implementation example 5
Clarity Clarification Clarification Clarification Clarification Clarification Clarification Clarification Clarification Clarification Clarification Clarification Clarification
From the result of table 8 as can be seen, no matter culture medium dry powder mixture of the present invention and culture media composition (liquid) are new systems or after storage for some time, can both reach the clarity with existing culture media composition par.
(2) pH value
According to two appendix VI of Pharmacopoeia of People's Republic of China version in 2005 H pH pH-value determination pH method, detect the culture medium dry powder composition of the Comparative Examples 1 of new system and embodiment 1-3 respectively and the culture medium dry powder composition of Comparative Examples 1 after storing 30 days under the environment of room temperature, relative humidity 50% and embodiment 1-3 mixes the medium liquid that the back forms with distilled water with pH meter, and the culture media composition (liquid) of the embodiment 4-5 after one week of preservation under 2 ℃ behind the culture media composition (liquid) of new system embodiment 4-5 and the filtration sterilization.For culture medium dry powder composition, get the consumption of 1 liter of preparation, place the 1000ml beaker, add water 1000ml, be stirred to dissolving; Directly test for culture media composition (liquid).Test result is as shown in the table:
Table 9
Sample Embodiment 1 Storage implementation example 1 Embodiment 2 Storage implementation example 2 Embodiment 3 Storage implementation example 3 Comparative Examples 1 Storage Comparative Examples 1 Embodiment 4 Storage implementation example 4 Embodiment 5 Storage implementation example 5
pH 4.8 4.9 5.0 5.1 5.3 5.3 4.3 4.4 5.3 5.3 5.5 5.5
From the result of table 9 as can be seen, no matter culture medium dry powder mixture of the present invention and culture media composition (liquid) are new systems or after storage for some time, can both reach the pH level identical, and the variation of pH is no more than 0.1 before and after culture medium dry powder mixture of the present invention and the culture media composition storage with existing culture media composition.
(3) loss on drying
According to two appendix VIII of Pharmacopoeia of People's Republic of China version in 2005 L dry weightless mensuration, detect the Comparative Examples 1 of new system and culture medium dry powder composition and Comparative Examples 1 after storing 30 days under the environment of room temperature, relative humidity 50% and the culture medium dry powder composition of embodiment 1-3 of embodiment 1-3 respectively.Test result is as shown in the table:
Table 10
Sample Embodiment 1 Storage implementation example 1 Embodiment 2 Storage implementation example 2 Embodiment 3 Storage implementation example 3 Comparative Examples 1 Storage Comparative Examples 1
Loss on drying (%) 3.0 3.1 2.5 2.4 3.2 3.2 3.4 3.5
From the result of table 10 as can be seen, no matter culture medium dry powder mixture of the present invention and culture media composition (liquid) are new systems or after storage for some time, can both reach the loss on drying level suitable, and the loss on drying variation before and after culture medium dry powder mixture of the present invention and the culture media composition storage is no more than 0.2% with existing culture media composition.
(4) osmotic pressure
According to two appendix IX of Pharmacopoeia of People's Republic of China version in 2005 G osmotic pressure molar density assay method, detect the culture medium dry powder composition of the Comparative Examples 1 of new system and embodiment 1-3 and the culture medium dry powder composition of Comparative Examples 1 after storing 30 days under the environment of room temperature, relative humidity 50% and embodiment 1-3 respectively and mixes the medium liquid that the back forms with distilled water, and the culture media composition (liquid) of the embodiment 4-5 after one week of preservation under 2 ℃ behind the culture media composition (liquid) of new system embodiment 4-5 and the filtration sterilization.For culture medium dry powder composition, get the consumption of 1 liter of preparation, place the 1000ml beaker, add water 1000ml, be stirred to dissolving; Directly test for culture media composition (liquid).Test result is as shown in the table:
Table 11
Sample Embodiment 1 Storage implementation example 1 Embodiment 2 Storage implementation example 2 Embodiment 3 Storage implementation example 3 Comparative Examples 1 Storage Comparative Examples 1 Embodiment 4 Storage implementation example 4 Embodiment 5 Storage implementation example 5
Osmotic pressure (mOsmol/ KgH 2O) 260 262 270 264 280 275 256 254 268 268 274 279
From the result of table 11 as can be seen, no matter culture medium dry powder mixture of the present invention and culture media composition (liquid) are new systems or after storage for some time, can both reach the osmotic pressure level identical, and the variation of osmotic pressure is no more than 10mOsmol/KgH before and after culture medium dry powder mixture of the present invention and the culture media composition storage with existing culture media composition 2O.
(5) bacterial endotoxin
According to the gel method in Pharmacopoeia of People's Republic of China version appendix in 2005 the XI E bacterial endotoxins test, detect the culture medium dry powder composition of the Comparative Examples 1 of new system and embodiment 1-3 and the culture medium dry powder composition of Comparative Examples 1 after storing 30 days under the environment of room temperature, relative humidity 50% and embodiment 1-3 respectively and mixes the medium liquid that the back forms with distilled water, and the culture media composition (liquid) of the embodiment 4-5 after one week of preservation under 2 ℃ behind the culture media composition (liquid) of new system embodiment 4-5 and the filtration sterilization.For culture medium dry powder composition, get the consumption of 1 liter of preparation, place the 1000ml beaker, add water 1000ml, be stirred to dissolving; Directly test for culture media composition (liquid).Get 0.1ml substratum (liquid) and add bacterial endotoxin inspection water (U.S. ACC) 3.9ml, mixing promptly gets testing liquid.Test result is as shown in the table:
Table 12
Sample Embodiment 1 Storage implementation example 1 Embodiment 2 Storage implementation example 2 Embodiment 3 Storage implementation example 3 Comparative Examples 1 Storage Comparative Examples 1 Embodiment 4 Storage implementation example 4 Embodiment 5 Storage implementation example 5
Bacterial endotoxin (EU/ml) ＜10 ＜10 ＜10 ＜10 ＜10 ＜10 ＜10 ＜10 ＜10 ＜10 ＜10 ＜10
From the result of table 12 as can be seen, no matter culture medium dry powder mixture of the present invention and culture media composition (liquid) are new systems or after storage for some time, can both be on close level with the bacterial endotoxin of existing culture media composition, meet of the requirement (＜10 EU/ml) of industry standard, and the variation of bacterial endotoxin level is no more than 1EU/ml before and after culture medium dry powder mixture of the present invention and the culture media composition storage to level of endotoxin.
(6) microbial limit
Undertaken by Pharmacopoeia of People's Republic of China version appendix in 2005 XI J limit test of microbe method, inspection item is bacterial count, fungi count, and test procedure is a Plating.For Zooblast culture medium dry powder composition, take by weighing 1g, be accurate to 0.01g, add axenic purification water 10ml dissolving, mixing can get testing liquid; Under aseptic condition, shift 10ml with directly as testing liquid for Zooblast culture medium (liquid) composition with the sterilization pipettor.Test result is as shown in the table:
Table 13
Sample Embodiment 1 Storage implementation example 1 Embodiment 2 Storage implementation example 2 Embodiment 3 Storage implementation example 3 Comparative Examples 1 Storage Comparative Examples 1 Embodiment 4 Storage implementation example 4 Embodiment 5 Storage implementation example 5
Microbial limit (CFU/g) Bacterium 50 50 50 50 50 50 50 50 50 50 50 50
Mould 0 0 0 0 0 0 0 0 0 0 0 0
From the result of table 13 as can be seen, no matter culture medium dry powder mixture of the present invention and culture media composition (liquid) are new systems or after storage for some time, can both be on close level with the microbial limit of existing culture media composition, meet of the requirement (bacteriums of＜200 CFU/gs) of industry standard, and the microbial limit level does not change before and after culture medium dry powder mixture of the present invention and the culture media composition storage to level of endotoxin.
(7) cell growth test
Cultivate embodiment 1
11022.4 milligrams of the Zooblast culture medium dry powder compositions that embodiment 1 is made are dissolved in the 900ml ultrapure water, add the 30ml calf serum and mix in the gained mixture, are settled to 1000ml with ultrapure water then.Use the bacteriological filtration membrane filtration sterilization of 0.20 μ m, promptly get nutrient solution.
Under aseptic condition, the above-mentioned nutrient solution of 10ml is transferred to 25cm with the sterilization pipettor 2In the Tissue Culture Flask, inoculation Vero cell suspension, inoculum density is 5 * 10 4Cell/ml.Cultivate under (37 ± 1) ℃ temperature condition and (5 ± 0.1) % carbonic acid gas volume score condition then, the area at the record unit fate cell covering bottle end and cell cover with the time at the bottom of the culturing bottle.Cultivation results is seen below civilian table 14, and 72 hours photo of cell cultures is seen Fig. 1.As seen from Figure 1, big by cultured cells density, the edge of cell is very clear, and form is normal.
Cultivate embodiment 2
13294.6 milligrams of the Zooblast culture medium dry powder compositions that embodiment 2 is made are dissolved in the 900ml ultrapure water, add the 40ml calf serum and mix in the gained mixture, are settled to 1000ml with ultrapure water then.Use the bacteriological filtration membrane filtration sterilization of 0.10 μ m, promptly get nutrient solution.
Under aseptic condition, the above-mentioned nutrient solution of 10ml is transferred to 25cm with the sterilization pipettor 2In the Tissue Culture Flask, inoculation Vero cell suspension, inoculum density is 5 * 10 4Cell/ml.Cultivate under (37 ± 1) ℃ temperature condition and (5 ± 0.1) % carbonic acid gas volume score condition then, the area at the record unit fate cell covering bottle end and cell cover with the time at the bottom of the culturing bottle.Cultivation results is seen below civilian table 14.
Cultivate embodiment 3
12316 milligrams of the Zooblast culture medium dry powder compositions that embodiment 3 is made are dissolved in the 900ml ultrapure water, add the 10ml calf serum and mix in the gained mixture, are settled to 1000ml with ultrapure water then.Use the bacteriological filtration membrane filtration sterilization of 0.15 μ m, promptly get nutrient solution.
Under aseptic condition, the above-mentioned nutrient solution of 10ml is transferred to 25cm with the sterilization pipettor 2In the Tissue Culture Flask, inoculation Vero cell suspension, inoculum density is 5 * 10 4Cell/ml.Cultivate under (37 ± 1) ℃ temperature condition and (5 ± 0.1) % carbonic acid gas volume score condition then, the area at the record unit fate cell covering bottle end and cell cover with the time at the bottom of the culturing bottle.Cultivation results is seen below civilian table 14.
Cultivate embodiment 4
In the Zooblast culture medium composition that 970ml embodiment 4 makes, add the 30ml horse serum and mix, use the bacteriological filtration membrane filtration sterilization of 0.15 μ m, promptly get nutrient solution.
Under aseptic condition, the above-mentioned nutrient solution of 10ml is transferred to 25cm with the sterilization pipettor 2In the Tissue Culture Flask, inoculation Vero cell suspension, inoculum density is 5 * 10 4Cell/ml.Cultivate under (37 ± 1) ℃ temperature condition and (5 ± 0.1) % carbonic acid gas volume score condition then, the area at the record unit fate cell covering bottle end and cell cover with the time at the bottom of the culturing bottle.Cultivation results is seen below civilian table 14.
Cultivate embodiment 5
Get the culture media composition 1000ml that embodiment 5 makes and directly use, wherein contain the calf serum of 2.9 volume %.
Under aseptic condition, the above-mentioned nutrient solution of 10ml is transferred to 25cm with the sterilization pipettor 2In the Tissue Culture Flask, inoculation Chinese hamster ovary celI suspension, inoculum density is 5 * 10 4Cell/ml.Cultivate under (37 ± 1) ℃ temperature condition and (5 ± 0.1) % carbonic acid gas volume score condition then, the area at the record unit fate cell covering bottle end and cell cover with the time at the bottom of the culturing bottle.Cultivation results is seen below civilian table 14, and 72 hours photo of cell cultures is seen Fig. 5.As seen from Figure 5, big by cultured cells density, the edge of cell is very clear, and form is normal.
Cultivate Comparative Examples 1
9513.11 milligrams of the Zooblast culture medium dry powder compositions that Comparative Examples 1 is made are dissolved in the 850ml ultrapure water, add the 100ml calf serum and mix in the gained mixture, are settled to 1000ml with ultrapure water then.Use the bacteriological filtration membrane filtration sterilization of 0.20 μ m, promptly get nutrient solution.
Under aseptic condition, the above-mentioned nutrient solution of 10ml is transferred to 25cm with the sterilization pipettor 2In the Tissue Culture Flask, inoculation Vero cell suspension, inoculum density is 5 * 10 4Cell/ml.Cultivate under (37 ± 1) ℃ temperature condition and (5 ± 0.1) % carbonic acid gas volume score condition then, the area at the record unit fate cell covering bottle end and cell cover with the time at the bottom of the culturing bottle.Cultivation results is seen below civilian table 14, and 72 hours photo of cell cultures is seen Fig. 2.As seen from Figure 2, big by cultured cells density, the edge clear of cell, form is normal.
Cultivate Comparative Examples 2
Adopt substratum [5% bovine serum+FGF-2 (10ng/ml)+Regular Insulin (10 μ g/ml)+PDGF (1ng/ml)+EGF (10ng/ml) dexamethasone (5 * 10 of embodiment 2 records of CN 1723277 -9M)+and zymoplasm (1 unit)], according to cultivating culturing cell under the identical condition of Comparative Examples 1, record unit fate cell covers the time at the bottom of the area at the bottle end and cell cover with culturing bottle.Cultivation results is seen below civilian table 14, and 72 hours photo of cell cultures is seen Fig. 3.As seen from Figure 3, big by cultured cells density, the edge clear of cell, form is normal.
Cultivate Comparative Examples 3
Prepare nutrient solution and culturing cell according to the method for cultivating Comparative Examples 1, different is only to add the 30ml calf serum.Cultivation results is seen below civilian table 14, and 72 hours photo of cell cultures is seen Fig. 4.As can be seen from Figure 4, because the serum that uses is few in the culturing process, cause that the Vero cell speed of growth is slow, density is low, the blur margin of cell is clear, and form is undesired.
Table 14
Animal serum addition (volume %) Inoculating cell density (cell/ml) 24 hourly average cell quantities (covering bottle floorage %) 48 hourly average cell quantities (covering bottle floorage %) 72 hourly average cell quantities (covering bottle floorage %) Time (h) at the bottom of being paved with bottle
Cultivate embodiment 1 3 5×10 4 50 85 100 60
Cultivate embodiment 2 4 5×10 4 60 90 100 60
Cultivate embodiment 3 1 5×10 4 40 70 90 75
Cultivate embodiment 4 3 5×10 4 50 83 100 72
Cultivate embodiment 5 2.9 5×10 4 50 82 100 72
Cultivate Comparative Examples 1 10 5×10 4 60 85 100 72
Animal serum addition (volume %) Inoculating cell density (cell/ml) 24 hourly average cell quantities (covering bottle floorage %) 48 hourly average cell quantities (covering bottle floorage %) 72 hourly average cell quantities (covering bottle floorage %) Time (h) at the bottom of being paved with bottle
Cultivate Comparative Examples 2 3 5×10 4 60 85 100 72
Cultivate Comparative Examples 3 3 5×10 4 30 60 80 90
From the result of table 14 as can be seen, Zooblast culture medium dry powder composition of the present invention and culture media composition can cooperate the low levels animal serum to use the animal serum of low levels (or contain), reach and use the high-content animal serum to maintain an equal level even better promote the effect of zooblast growth.Owing to adopt Zooblast culture medium dry powder composition of the present invention and culture media composition, in the whole process of animal cell culture, under the prerequisite that does not influence the cell growth, the usage quantity of serum can significantly reduce, thereby the cost of scale operation is significantly reduced.Because Zooblast culture medium dry powder composition of the present invention need not additionally to introduce protein ingredient (for example recombinant protein, vegetable-protein or animal-origin component such as cytokine etc.), therefore do not produce the side effect of following protein ingredient, security is good.

Claims (15)

1. a Zooblast culture medium dry powder composition is characterized in that, this Zooblast culture medium dry powder composition comprises the component shown in the following table 1:
Table 1
Component Weight part Component Weight part The L-L-Ala 80-120 Calcium nitrate tetrahydrate 30-80 The L-arginine monohydrochloride 180-210 Repone K 320-500 The L-aspartic acid 5-30 Anhydrous magnesium sulfate 40-80 The L-asparagine 30-60 Sodium-chlor 6000-8000 The L-cysteine hydrochloride 10-30 Disodium hydrogen phosphate,anhydrous 300-400 L-L-glutamic acid 100-150 Iron vitriol 0.1-1 L-glutaminate 200-500 Zinc vitriol 0.2-3 The L-L-Histidine hydrochloride 20-50 D-glucose 1000-3000 The L-oxyproline 2-15 Gsh 0.2-2 The L-Isoleucine 90-150 Sodium.alpha.-ketopropionate 20-120 The L-leucine 10-70 Xitix 1-10 L lysine HCL 100-200 Choline chloride 60 5-25 The L-methionine(Met) 10-50 The D-calcium pantothenate 1-10 The L-phenylalanine 10-50 Folic acid 2-10 The L-proline(Pro) 20-60 Niacinamide 1-5 The L-Serine 20-60 Pyridoxine hydrochloride 1-10 The L-Threonine 60-100 Riboflavin 0.2-2 The L-tryptophane 10-50 Thiamine hydrochloride 1-10 The L-Xie Ansuan 30-70 Vitamin B12 1-10 L-tyrosine 30-80 Magnesium dichloride hexahydrate 40-100
Component Weight part Component Weight part Glycine 30-80 Xanthoglobulin 1-10 The L-cystine hydrochloride 30-80 Thymidine 0.1-2 Calcium Chloride Powder Anhydrous 30-80 Anhydrous potassium dihydrogenphosphate 20-100 AMSP 30-80
2. Zooblast culture medium dry powder composition according to claim 1, wherein, described Zooblast culture medium dry powder composition comprises the component shown in the following table 2:
Table 2
Component Weight part Component Weight part The L-L-Ala 90-110 Calcium nitrate tetrahydrate 30-60 The L-arginine monohydrochloride 190-210 Repone K 320-400 The L-aspartic acid 5-20 Anhydrous magnesium sulfate 50-70 The L-asparagine 30-50 Sodium-chlor 6500-7500 The L-cysteine hydrochloride 10-15 Disodium hydrogen phosphate,anhydrous 300-380 L-L-glutamic acid 100-120 Iron vitriol 0.1-0.5 L-glutaminate 300-500 Zinc vitriol 0.2-1.5 The L-L-Histidine hydrochloride 30-50 D-glucose 2000-3000 The L-oxyproline 2-10 Gsh 0.6-1.5 The L-Isoleucine 90-120 Sodium.alpha.-ketopropionate 50-100 The L-leucine 30-70 Xitix 2-7 L lysine HCL 100-180 Choline chloride 60 10-16 The L-methionine(Met) 20-50 The D-calcium pantothenate 2-6 The L-phenylalanine 30-50 Folic acid 3-6 The L-proline(Pro) 40-60 Niacinamide 2-4
Component Weight part Component Weight part The L-Serine 40-60 Pyridoxine hydrochloride 2-6 The L-Threonine 60-80 Riboflavin 0.5-1 The L-tryptophane 10-30 Thiamine hydrochloride 2-5 The L-Xie Ansuan 40-70 Vitamin B12 3-7 L-tyrosine 30-50 Magnesium dichloride hexahydrate 50-80 Glycine 30-60 Xanthoglobulin 1-4 The L-cystine hydrochloride 40-60 Thymidine 0.5-1 Calcium Chloride Powder Anhydrous 60-80 Anhydrous potassium dihydrogenphosphate 50-90 AMSP 40-70
3. Zooblast culture medium dry powder composition according to claim 1 and 2, wherein, described cell dry powder composite also comprises one or more in the vitamin K3 of vitamin D2,1-3 weight part of linolic acid, the 1-3 weight part of 1-3 weight part and 2-10 weight part phenol red.
4. the preparation method of a Zooblast culture medium dry powder composition, this method comprises that the component that will be selected from any described Zooblast culture medium dry powder composition among the claim 1-3 is dispersed in the dispersion agent, removes the dispersion agent in the gained mixture.
5. method according to claim 4 is characterized in that, one or more in alcohol, alkaline aqueous solution and the acidic aqueous solution that described dispersion agent is selected from distilled water, deionized water, water for injection, redistilled water, ultrapure water, can dissolve each other with any ratio with water.
6. method according to claim 5, wherein, described alkaline aqueous solution is the aqueous solution of the cationic alkali that occurs in the Zooblast culture medium dry powder composition described in sodium hydroxide solution or the claim 1-3, and described acidic aqueous solution is the aqueous acid of the acid ion that occurs of the Zooblast culture medium dry powder composition described in the claim 1-3.
7. Zooblast culture medium composition, said composition comprises Zooblast culture medium dry powder composition and supporting agent thereof, it is characterized in that described Zooblast culture medium dry powder composition is selected from any described Zooblast culture medium dry powder composition among the claim 1-3.
8. Zooblast culture medium composition according to claim 7, wherein, described supporting agent is selected from one or more in distilled water, deionized water, water for injection, redistilled water and the ultrapure water.
9. Zooblast culture medium composition according to claim 8, wherein, described supporting agent is water for injection and/or ultrapure water.
10. Zooblast culture medium composition according to claim 7, wherein, the consumption of described supporting agent is the 600000-1500000 weight part.
11. Zooblast culture medium composition according to claim 10, wherein, the consumption of described supporting agent is the 800000-1200000 weight part.
12. Zooblast culture medium composition according to claim 11, wherein, the consumption of described supporting agent is 1000000 weight parts.
13. Zooblast culture medium composition according to claim 7, wherein, described Zooblast culture medium composition comprises that also final concentration is that 1 volume % is to the animal serum less than 10 volume %.
14. Zooblast culture medium preparation of compositions method, this method comprises Zooblast culture medium dry powder composition is dispersed in the supporting agent, filtration sterilization, it is characterized in that described Zooblast culture medium dry powder composition is selected from one or more in the Zooblast culture medium dry powder composition described in the claim 1-3.
15. Zooblast culture medium preparation of compositions method according to claim 14, this method comprise that also adding final concentration in the supporting agent that is dispersed with described culture medium dry powder composition is that 1 volume % is to the animal serum less than 10 volume %.
CN2007101217097A 2007-09-12 2007-09-12 Zooblast culture medium dry powder composition, culture medium composition and preparation method thereof Expired - Fee Related CN101386836B (en)

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