CN1470632A - Optimized feeding suspension culture method for ammal cell - Google Patents

Abstract

The present invention relates to an optimum flow and suspension culture method of animal cell, which utilizes the research of cell growth kinetics to define the minimum concentration of nutrient material required for cell growth, and utilizes the chemical metering method to define and synthesize a proportion of the related nutrient materials required for cell growth, and uses that as basis to design a reasonable formula of all nutrient materials adding nutrient material flow, and said formula can be used for controlling chemical environment for cell growth. Said invention features high application efficiency of culture solution, high cell density, high protein concentration, high yield and low cost, etc.

Description

A kind of optimization stream of zooblast adds the suspension culture method

Technical field

The present invention relates to a kind of zooblast be carried out the high-concentration suspension cultured method, or rather, the optimization stream that the present invention relates to a kind of zooblast adds the suspension culture method.

Background technology

The animal cell culture technology just begins (Eagle is needs of mammalian cells in tissue culture.Science 122:501-504. H.1955.Nutrition) as far back as the beginning of the fifties, through the development of decades, cell culture technology has obtained good application in the production of pharmaceutical grade protein and virus vaccines and gene therapy product.

It is a kind of comprehensive, multi-disciplinary and highly difficult sophisticated technology and art that high-density animal cell is cultivated.Obtain supply and control, the control that toxic byproduct is arranged or the discharge that high cell density need solve the required nutrition of cell metabolism, the supply and the environmental parameter of oxygen, as the pH value, osmotic pressure (osmolarity), oxyty (dissolved oxygen), problems such as the real-time control of temperature etc.Solve unit operation and the special knowledge and the technical ability such as technological process control and optimization of biochemical reaction system that these technical problems need to be grasped cell, cells physiological, chemical engineering.

The purpose that the research high-density animal cell is cultivated is the protein output for the nutrient solution that improves unit reactor volume and unit volume, thereby reaches high efficiency, low fixed capital (plant and equipment) drop into and lower production cost.Improving the concentration and the output of proteinaceous product in the animal cell culture must set about from three aspects: improve the protein expression level in the host cell; Improve cell concn; With the prolongation cell cultures life-span.

The expression level of monoclonal antibody in host cell is a crucial processing parameter, and it directly influences the concentration and the output of monoclonal antibody.Under normal conditions, the protein expression level is 10 * 10 ^-12About gram/cell/sky.Protein expression level in the host cell depends on the feature of gene aspect, structure, gene copy number and the stability of alien gene in host cell as the protein expression carrier, with cell growing environment factor, as the concentration of the saturating pressure (osmolarity) of the speed of growth of cell, nutrient solution, Sodium propanecarboxylate (sodium butyrate) and cell cultures temperature etc.Optimize the protein expression carrier structure, can directly improve proteinic output.Further improve physics, chemical environment that the antibody expression level need increase the copy number of antibody gene and optimize cell cultures.General in the world increase copy number of target genes purpose technology has two classes: at DHFR ^-The Chinese hamster ovary celI system adopts to be increased Methotrexate (methotrexate (MTX)) concentration and adopts increase methionine(Met) sulphur ammonia (methionine sulfoxamine) concentration [Bebbington CR at GS-NSO (glutamine synthetase) cell system, RennerG, Thomson S, King D, Abrams D, Yarranton GT.1992.High-levelexpression of a recombinant antibody from myeloma cells using aglutamine synthetase gene as an amplifiable selectable marker.Biotechnology 10:169-175; Robinson DK, DiStefano D, Gould SL, CucaG, Seamans TC, Benincasa D, Munshi S, Chan CP, Stafford-Hollis J, Hollis GF, Jain D, Ramasubramanyan K, E.MG, Silberklang is of Engineered Antibodies in Myeloma and Hybridoma Cells:Enhancements in Gene Expression and Process Design.AntibodyEngineering.H.Wang and T.Imanaka.Worthington M.1995.Production, ACS.; YoshikawaT, Nakanishi F, Ogura Y, Oi D, Omasa T, Katakura Y, Kishimoto M, SugaK.2000.Amplified gene location in chromosomal DNA affectedrecombinant protein production and stability of amplified genes.Biotechnology ﹠amp; Bioengeering 16:710-715.].The concentration that increases the inhibitor of cell growth is unfavorable for the cell growth of the copy number of antibody gene low (thereby its DHFR or GS gene copy number also low), thereby obtains to contain the cell strain of the copy number height of eye of antibody gene selectively.Adopt this method, Robinson et al.[1995] successfully with the antibody expression level before optimize 10 ^-115 * l0 is brought up in gram/cell/sky ^-11Gram/cell/sky, the copy number of antibody gene is also corresponding brings up to 5 from original 1.

Another method that improves protein expression level is to optimize the environment of cell growth: comprise temperature, pH value, saturating pressure and add chemical substance such as the Sodium propanecarboxylate that some can the irritation cell protein expression.Chang et al.[Chang DYH.2000.Development of a technologyplatform for production of human monoclonal antibodies in recombinantCHO cell culture.Cell Culture Engineering Conference VII.] by improving the chemical environment of cell cultures, successfully with the expression level of monoclonal antibody in Chinese hamster ovary celI from 2.1 * 10 ^-11Gram/cell/sky brings up to 3.5 * 10 ^-11Gram/cell/sky.Oh et al.[Oh SKW, Vig P, Chua F, Teo WK, Yap MGS.1993.Substantial overproduction ofantibodies by applying osmotic pressure and sodium butyrate.Biotechnology ﹠amp; Bioengeering 42:601-610.] by in nutrient solution, adding Sodium propanecarboxylate and improving saturating way of pressing and improve the protein expression level.The environmental parameter that visible cell is cultivated is to the material impact of protein production.

Another important method that improves protein output is the growth time that improves cell density and prolong cell, adopts stream to add the technology process and can obtain good effect.Stream adds the technology that the technology process is a kind of batch production basically, different is in cell cultivation process, the employing reinforced mode of getting over is replenished the nutrition that is consumed in the cell growth process, with this time that prolongs the cell growth, reaches high cell density.Common way is to adopt gyp batch production nutrient solution, carry out batch production earlier, soon near the coda of batch production the time, the mixture of the nutritive substance that adding is prepared in advance in reactor [Park S, Ramifez WF.1988.Optimal productionof Secreted protein in fed-batch reactors.AIChE is J.34:1550-1558.; Tremblay Md, Perrier M, Chavarie C, Archambault be fed-batch culture of hybridom cells using dynamic programming:single and multi feed cases.Bioprocess Engineering 7:229-234 J.1992.Optimizationof].Since tens kinds of nutritive substances of cell growth needs, and reinforced logistics has only one usually, determines that the composition of reinforced logistics just becomes very important, but lack a kind of system reasonable method at present.Common way is to cultivate spissated reinforced logistics according to gyp batch production with the composition of nutrient solution, its shortcoming is that the material that has too much causes accumulation, some material causes inadequately and exhausts too early and necrocytosis [Bushell ME, Bell SL, Scott MF, Spier RE, Wardell JN, SandersPG.1994.Enhancement of monoclonal antibody yield by hybridomfed-batch culture, resulting in extended maintenance of viable cellpopulation.Biotechnology ﹠amp; Bioengeering 44:1099-1106.; Jo E, ParkH, Kim D, Moon HM.1993.Repeated fed-batch culture of hybridoma cellsin nutrient-fortified high density medium.Biotechnol.Bioeng.42:1229-1237.].

Summary of the invention

The optimization stream that the invention provides a kind of zooblast adds the suspension culture method, may further comprise the steps:

A). determine the minimum concentration of cell growth desired nutritional material, be made into an initial nutrient environment that carries out cell cultures, will treat that culturing cell is inoculated in this initial nutrient environment;

B). provide one or more varying parameters, determine the ratio of synthetic needed all the crucial nutritive substances of cell in view of the above, and be made into reinforced logistics with this; Press the wear rate of nutritive substance in the nutrient environment, the logistics of will feeding in raw material adds in the nutrient environment, carries out cell cultures;

C). measure the varying parameter of actual culture system; The varying parameter value of this actual measurement is updated to b) in the step;

D). repeat b) and c) step one or many, try the difference iteration, reach to optimize and no longer be changed to up to nutrient environment and end.

Determine and the best nutritional environment of control cell growth by the present invention, thereby apoptosis is reduced and delay, obtaining high-cell density and to prolong the cell cultures time, and obtain high protein concn and output.The present invention determines the best nutritional environment that cell is grown by the method for research growth kinetics of cells, satisfies the required nutrition quality of cell growth and add technology by stoichiometric relation and stream.Thereby solved the growth kinetics of cells conflict different to the nutritive substance concentration requirement with stoichiometric relation.This invention can reduce the generation of Toxic waste, does not influence cell growth rate, can obtain high cell density again.Specify as follows:

Zooblast can produce deleterious refuse such as ammonia and lactic acid in process of growth, they are main by products of glucose and L-glutamic acid.Studies show that, reduce the output of ammonia and lactic acid effectively, must ease down to glucose and L-glutamic acid below the 1mM.But then, the growth of cell need consume a large amount of nutritive substances, especially glucose and L-glutamic acid.The present invention adopts a kind of novel stream to add the technology process to reach this two purposes simultaneously effectively: adopt that prepared culture is as the initial nutrient environment of cell growth voluntarily, its crucial nutrient concentrations is determined by growth kinetics of cells institute; Adopt the interpolation speed of controlling reinforced logistics to replenish the nutritive substance of cell consumption, the initial environment of cell growth is remained unchanged, the nutritional formula of reinforced logistics is determined by stoichiometric relation.

By method of the present invention, can adopt the growth kinetics of cells method to determine the minimum concentration of the nutritive substance that the cell growth is required.The research method of growth kinetics of cells early has document to deliver [Glacken MW, Adema E, Sinskey AJ.1988.Mathematical descriptions ofhybridoma culture kinetics:I.initial metabolic rates.Biotechnology﹠amp; Bioengeering 32:491-506.; Hayter PM, Curling EMA, Baines AJ, Jenkins N, Salmon I, Strange PG, Bull AT.1991.Chinese hamster ovarycell growth and interferon production kinetics in stirred batchculture.Applied Microbiology Biotechnology 34:559-564.; Miller WM, Blanch HW, Wilke CR.1988.A kinetics analysis of hybridoma growthand metabolism in batch and continuous suspension culture:effectof nutrient concentration, dilution Rate, and pH. Biotechnology ﹠amp; Bioengeering 32:947-965.; Ozturk SS, Palsson BO.1991.Growth, metabolic, and antibody production kinetics of hybridoma cell culture:1.analysis of data from controlled batch reactors.BiotechnologyProgress 7:471-480.].Incorporate related content into the application in this statement, for reference.The purpose of research cell growth is often in order to set up the mathematical model of calculating cell growth rate.The present invention then determines the required minimum nutrient matter concentration of cell growth by the research growth kinetics of cells, thereby makes the generation speed of Toxic waste drop to minimum.After determining the required minimum nutrient matter concentration of cell growth by growth kinetics of cells, be made into an initial nutrient environment that carries out cell cultures, and then will treat that cultured cells is inoculated in this initial nutrient environment.

By method of the present invention, can adopt stoechiometric process to determine the ratio of the used crucial nutritive substance that a synthetic cell is required.Dominate the research of the stoichiometric relation of cell growth and deliver [Xie L, Wang DIC.1994a.Stoichiometric analysis of animal cellgrowth and its application of medium design.Biotechnology ﹠amp; Bioengeering 43:1164-1174.; Xie L, Wang DIC.1994b.Fed-batchcultiVation of animal cells using different medium design conceptsand feeding strategies.Biotechnology ﹠amp; Bioengeering 43:1175-1189.; Xie L, Wang DIC.1994c.Applications of improved stoichiometric modelin medium design and fed-batch cultivation of animal cells inbioreactor.Cytotechnology 15:17-29.].Incorporate related content into the present patent application in this statement, for reference.Be summarized as follows:

Each zooblast all is a very complicated organism, also can be regarded as a machine of reproduction voluntarily.Zooblast contains unnumbered various organic compound, but roughly can be divided into following several big classification: protein (protein), sugary carbohydrates (carbohydrates), lipoid substance (lipids), DNA, RNA and other materials.Cell must synthetic cell in the growth and the process of self-replacation in all compounds.Obviously, must carry out ten hundreds of different chemical reactions in the cell, thereby we can say that cell itself is exactly a multicomponent reaction device that has a lot of chemical reactions to carry out simultaneously.More complicated is that under different ambient environmental conditions, cell can select different means of chemical reaction to synthesize some compounds.Obviously, the reactor of such complexity can't be described with one group of mathematical equation, and because its polytropy, such reactor assembly does not have unique solution.

By the characteristic of actual problem of being concerned about in the research cell cultivation process and cell growth, the inventor finds, although the metabolic mode of cell has mutability, to some specific cell strains, must have one best mode.It is optimum that the metabolic efficiency of the growth of cell with this understanding,, proteinic production and cell etc. all reaches.In this supposition ideally, unnecessary side reaction can not consider that the situation of multiple reaction path only need be considered wherein best, thereby the chemical reaction number that needs to consider significantly reduces.Then, again according to the classification of essential substance in the cell with the processing of concluding, classify of related chemical reaction, obtain one and comprise a plurality of equational chemical reaction network systems.Adopt the cell culture condition of a setting, utilize the matter and energy equilibrium relationship can solve this reaction network system, thereby obtain a chemical equation that can be described in cell growth process best under the culture condition of setting:

θ wherein _GlcFor glucose (glucose), θ _{A, i}For amino acid (amino acids), θ _{V, i}For VITAMIN (Vitamins), θ _pFor proteinaceous product (product), θ _ATPFor the required energy of cell metabolism (ATP), θ _kStoichiometric coefficient for by product (by-products).

Determine that these coefficients need measure the content of intracellular chemical substance and the required energy of cell growth, comprising total content and their amino acid whose average composition, the content of all sugary carbohydrates (carbohydrates), the content of all lipoid substances (lipids) and the total content of DNA and RNA of all proteins in the cell.In addition, also need to calculate the minimum amount of by-products of required energy of cell growth and designing institute permission.

Because the growth of cell to the consumption of the nutritive substance in the bio-reactor, must compensate the nutritive substance that consumes, just can make the nutrient environment of cell growth maintain initial optimum regime.According to the chemical reaction engineering principle, the ratio of the chemical ingredients in the feed stream should be mated with the reactive chemistry quantitative relation that is carried out, just can make the feeding rate and the quiet wear rate balance of this material in reactor of each material, the concentration of the chemical substance in the controlling reactor is constant effectively.According to the optimum chemical metering mathematical model of this ultimate principle and above dominant force cell growth of setting up, the molar fraction X of chemical substance i in the feed stream _iShould be directly proportional with its stoichiometric coefficient: $X_i=\frac{{\mathrm{\θ}}_i}{{\mathrm{\Σ\θ}}_i}...........\left(2\right)$

θ wherein _iBe the stoichiometric coefficient of chemical substance i, ∑ θ _iStoichiometric coefficient summation for all chemical substances in the feed stream; The feed rate of feed stream then is:

Feed rate=N _t∑ θ _i(3)

N wherein _tBe the rate of rise of cell number, can determine according to the above growth kinetics of cells equation of studying.

Adopt above method to control the concentration of all nutritive substances in bio-reactor effectively, thereby prevent the accumulation of some material and exhausting of other material with the feed rate of a feed stream.

Owing to be cell culture condition first a setting, as the Toxic waste of supposing a permission produces under the speed (adjustable parameter), find the solution the reaction network system that institute's merger forms, thereby set up equation (1), (2) and (3), one is issued to the best nutritional environment so the stream that is made into by equation (1), (2), (3) during beginning adds that material is formed and the stream dosage can not guarantee that nutrient environment that cell is grown does not change.Therefore need find the solution parameter in the formula (2) again according to the actual cell growing nutrient environment that measures, redefine the ratio of synthetic needed all key substance of cell under New Terms in formula (2), adjustable parameter in the stoichiometry model is carried out necessary correction, be made into reinforced logistics, and determine feed rate by formula (3).This process is called iterative process one time.Whenever carry out obtaining a new nutrient environment after an iteration, thereby revise parameter in expression of first degree (1), (2) and (3), after adjusting stream in view of the above and adding the composition of material and flow dosage, finish a new iterative process again.Repeat this iterative process one or many, reach optimization, till promptly minimum the and nutrient environment of the generation speed of Toxic waste no longer changes up to nutrient environment.

By the preferred embodiment of the inventive method, used zooblast is any in the cell that is selected from CHO, hybridoma, PER.C6,293, the insect cell.

The present invention is suitable for the vitro culture of various suspension mammalian cells, comprises CHO, hybridoma (SP2/0, NSO, etc.), PER.C6,293, HeLa, insect cell etc.Can in various different cultivation scales (laboratory and industrial scale) and reactor (shake bottle, rolling bottle, stir and block reactor etc.), use.

By a preferred embodiment of method of the present invention, used initial incubation liquid is serum-free.

By a preferred embodiment of the inventive method, the feed way that stream adds material can be manual or automatization.

Description of drawings

Fig. 1 is the growing state contrast of Chinese hamster ovary celI when cultivating at batch process with by the inventive method.

Growing state when wherein curve 1 is cultivated for press batch process, curve 2 are the growing state when cultivating by the inventive method.

Embodiment

As a kind of example to the inventive method, this be described in detail a kind of in conjunction with growth kinetics of cells research and stoichiometric relation and adopt zooblast that iteration is optimized to optimize stream to add the suspension culture Technology as follows.

1. determine the best nutritional environment of cell growth:

Adopt prepared culture and batch cell strain that the technological process cultivation will be studied voluntarily, obtain the fundamental characteristics of cell growth.In physical parameter (temperature, pH value, oxyty etc.) be subjected under the condition of strict control, carry out following contrast experiment: nutrient solution that does not contain glucose of (1) preparation adds different glucose concn then (as 0.1,0.5,1.0,2.0,5.0,10.0,25.0mM), and be used for culturing cell and measure growth velocity and the poisonous production of by-products speed of cell under different glucose concn; (2) nutrient solution that does not contain L-glutamic acid of preparation adds different aminoglutaric acid concentrations then (as 0.1,0.2,0.4,0.6,1.0,2.0,4.0mM), and be used for culturing cell and measure growth velocity and the poisonous production of by-products speed of cell under different aminoglutaric acid concentrations; (3), determine not influence the minimum glucose and the aminoglutaric acid concentration of cell growth rate, and prepare stream with this and add the used initial incubation liquid of technology according to above gained data.

2. determine the stoichiometric relation of cell growth:

Adopt the method for having delivered to measure the dry weight of cell and content (the Xie and Wang of cell inner macromolecule material, 1994c), suppose that one allows Toxic waste to produce speed (adjustable parameter), according to the stoichiometric coefficient of the mathematical model of having delivered and method calculating glucose, amino acid and VITAMIN.Calculate the nutritive substance prescription of reinforced logistics according to equation (2), and prepare reinforced logistics.

3. adopt the stream of stoichiometry control to add technology and carry out animal cell culture:

Adopt the initial nutrient environment inoculating cell of institute's prepared culture conduct in the step 1, the beginning cell cultivation process.The interpolation speed of the reinforced logistics of preparation and the reinforced logistics of equation (3) control realizes the control of the nutrient environment of cell growth in the employing step 2.Measure the parameter in the cell growth process, produce speed, lactic acid generation speed and glucose and aminoglutaric acid concentration variation etc. as growth velocity, ammonia.

4. the experimental result according to step 3 is optimized adjustable parameter:

Ammonia according to above step gained produces speed and lactic acid generation speed and experimental datas such as glucose and aminoglutaric acid concentration variation, adjustable parameter in the stoichiometry model (the generation speed of ammonia and lactic acid) is carried out necessary correction, promptly adopt the ammonia of measuring in the step 3 to produce speed, lactic acid generation speed, recomputate the stoichiometric coefficient of cell growth, and prepare new reinforced logistics with this.Stream in the repeating step 3 adds the technology culturing process, reaches the minimum stream that is optimization up to the generation speed of ammonia and lactic acid and adds Technology.

The present invention is suitable for the vitro culture of various suspension mammalian cells, comprises CHO, hybridoma (SP2/0, NSO, etc.), PER.C6,293, HeLa, insect cell etc.Can in various different cultivation scales (laboratory and industrial scale) and reactor (shaking bottle, rolling bottle, stirred reactor etc.), use.

Example one

Experimental technique: this experiment adopts the genetically engineered Chinese hamster ovary celI strain of expressing a monoclonal antibody to experimentize.Used initial incubation liquid is the serum-free medium of independent development, wherein contains the glucose of 3.0mM and the L-glutamic acid of 0.5mM, and other amino acid whose concentration commercial nutrient solution also commonly used decreases.Test under the speed that the rolling bottle (Spinner Flask) that adopts 500 milliliters is per minute 150 commentaries on classics at rotating speed and carry out, the Chinese hamster ovary celI inoculum density is 0.4 * 10 ⁶Cells/ml, temperature are controlled to be 37 ℃.The prescription of initial incubation liquid sees Table one, and the prescription of reinforced logistics sees Table two.

Experimental result: figure one has shown Chinese hamster ovary celI under the serum-free culture condition, when adopting common batch of technology and adding technology by optimization stream of the present invention, and the contrast of cell growing state and cell density.In batch technology was cultivated, nutrient concentrations was similar with commercial nutrient solution commonly used in the serum-free medium, although also have a large amount of nutritive substance residues, high-cell density only is 3 * 10 ⁶Cells/ml; In contrast be, adopt stream of the present invention to add the technology process through the optimization of very short time, reached 3 * 10 ⁶Cells/ml.Proteinic output is also corresponding to have improved three times.It should be noted that these results obtain in the rolling bottle that lacks automatization control is cultivated, can infer, in the bio-reactor of control automatically, will have better result.Table one: the serum-free culture liquid formula that Chinese hamster ovary celI is cultivated

The component mg/litre	The component mg/litre
		Calcium dichloride dihydrate (CaCl 22H 2O) 147 sal epsom (MgSO 4) 72 potassium oxides (KCl), 298 sodium bicarbonate (NaHCO 3) 2940 sodium-chlor (NaCl), 4091 SODIUM PHOSPHATE, MONOBASIC (NaH2PO 4) 240 cupric sulfate pentahydrate (CuSO 4*5H 2O) 0.00075 sodium selenate (Na 2SeO 3) 0.0173 7 water zinc phosphate (ZnSO 4*7H 2O ) 0.2 ( Arginine ) 87 ( Asparagine ) 75 ( Aspartic Acid ) 53 ( Cysteine ) 36 ( Glutamic Acid ) 44 ( Glutamine ) 71 ( Glycine ) 37.5 ( Histidine ) 83.8 ( Isoleucine ) 105 ( Leucine ) 105 ( Lycine ) 146 ( Methionine ) 60 ( Phenylalanine ) 66 ( Proline ) 46 ( Serine ) 53 ( Threonine ) 71.5 ( Tryptophan ) 61 ( Tyrosine*2Na ) 105 ( Valine ) 70 ( D-Glucose ) 540	HEPES 6508 ( Pyruvate.Na ) 110 H ( D-Biotin ) 0.013 ( Choline Chloride ) 10 ( myo-Inositol ) 10 ( Niacinamide ) 3 ( D-Ca Pantothenate ) 3 ( Pyridoxal ) 3 ( Thiamine ) 3 B-12 ( Vitamin B12 ) 0.3 G ( Riboflavin ) 0.3 BC ( Folic Acid ) 3 ( Linoleic Acid ) 0.075 ( Lipoic Acid ) 0.2 ( Putrescine ) 0.16 EDTA 5.84 ( Cholesterol ) 0.1 ( glutathione ( reduced ) ) 0.5 C ( ascorbic acid ) 0.5 80 ( tween 80 ) 0.2 ( LiCl ) 0.02 ( NH4VO 3) 0.0003 Sodium orthomolybdate (Na 2MoO 4) 0.00035 nickelous chloride (NiCl 2) 0.0001 protein degradation thing (Primatone RL) 2000 Regular Insulin (Insulin), 2 phenolsulfonphthaleins (Phenol Red) 5.3

Table two: the prescription of reinforced logistics is formed

Composition	Concentration (grams per liter)
		Histidine (Arginine)	????2.32
Halfcystine (Cysteine)	????0.76
		Histidine (Histidine)	????1.02
Isoleucine (Isoleucine)	????1.25
		L-LEU (Leucine)	????2.39
Methionin (Lysine)	????2.79
		Methionine(Met) (Methionine)	????0.73
Phenylalanine (Phenylalanine)	????1.18
		Serine (Serine)	????2.13
Threonine (Threonine)	????1.54
		Tryptophane (Tryptophan)	????0.50
A word used in person's names propylhomoserin (Valine)	????1.6
		Glucose (D-glucose)	????59
SODIUM PHOSPHATE, MONOBASIC (NaH 2PO 4)	????2.22
		Aspartic acid (Aspartic acid)	????2.05
Sodium L-tyrosinate (Tyrosine.2Na)	????1.06
		Vitamin H (D-Biotin)	????0.023
Choline chloride 60 (Choline Chloride)	????0.64
		Inositol (Myo-Inositol)	????0.44
Nicotinoyl acid (Niacinamide)	????0.081
		Pyridoxal (Pyridoxal)	????0.19
VitB1 (Thiamine)	????0.051
		Pantothenic acid (D-Pantothenic acid)	????0.046
Vitamin B-12 (Vitamin B-12)	????0.016
		Vitamin(e) G (Riboflavin)	????0.022
Folie Acid (Folic acid)	????0.074
		Linolic acid (Linoleic acid)	????0.005
Thioctic Acid (Lipoic acid)	????0.001
		Thanomin (Etanolamine)	????0.01
Gsh (Glutathione (reduced))	????0.001
		Putrescine (Putrescine)	????0.0016
N (Asparagine)	????0.0016
		Ancient acyl ammonia (Glutamine)	????14.9

Claims (6)

1. the optimization stream of a zooblast adds the suspension culture method, may further comprise the steps:

A). determine the minimum concentration of cell growth desired nutritional material, be made into an initial nutrient environment that carries out cell cultures, will treat that culturing cell is inoculated in this initial nutrient environment;

B). provide one or more varying parameters, determine the ratio of synthetic needed all the crucial nutritive substances of cell in view of the above, and be made into reinforced logistics with this; Press the wear rate of nutritive substance in the nutrient environment, the logistics of will feeding in raw material adds in the nutrient environment, carries out cell cultures;

C). measure the varying parameter of actual culture system; The varying parameter value of this actual measurement is updated to b) in the step;

D). repeat b) and c) step one or many, try the difference iteration, reach to optimize and no longer be changed to up to nutrient environment and end.

2. by the method for claim 1, it is characterized in that, at b) in the step, determine the ratio of synthetic needed all the crucial nutritive substances of cell in stoechiometric process.

3. by the method for claim 2, it is characterized in that, in a) going on foot, adopt the growth kinetics of cells method to determine the minimum concentration of cell growth desired nutritional material.

4. by the method for claim 3, it is characterized in that described zooblast is to be selected from Chinese hamster ovary celI, hybridoma, a kind of in PER.C6,293, the insect cell.

5. by each method among the claim 1-4, it is characterized in that used initial incubation liquid is serum-free.

6. by the method for claim 5, it is characterized in that feed way is manual or automatization.

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