CN1772883A - Animal cell feeding culture process - Google Patents

Abstract

The animal cell feeding culture process includes the following steps: designing different feeding processes to realize different pseudostable culture environment, determine optimized target and establish metabolism dynamics model; performing the optimized culture process based on the metabolism dynamics model; sampling and detecting ammonia concentration regularly, estimating the concentration of glutamine as the control quantity based on the metabolism dynamics model and feeding back to correct feeding rate to avoid system divergency. The process of the present invention can perform research on metabolism regulations of cell strain fast and accurately and realize cell culture process with high culture medium efficiency, high cell density and product concentration, high stability and controllability. The present invention makes it possible to establish large scale optimized animal cell feeding culture process and is significant in producing bioproduct.

Description

Animal cell feeding culture process

Technical field

Extensive, the hyperpycnal flow that the present invention relates to a kind of zooblast add cultured method.

Technical background

Animal cell large-scale is cultivated and has been widely used in producing all kinds of biologically active substances such as monoclonal antibody, virus vaccines, immune-regulating factor, somatomedin, particular tumor antigens and range gene recombinant protein drug.Zooblast is compared the ability with post transcriptional modificaiton with microorganism, can express and produce all kinds of high-quality protein efficiently.Yet the substratum that is used for animal cell culture costs an arm and a leg, and expression amount is low to cause the cost of separation and purification higher.Therefore, improve cell density and production concentration, it is significant on industrial application to reduce substratum consumption.

Cultivate in (Batch) for simple batch, along with nutraceutical consumption and byproducts build-up, cell stops growing and begins death, and product also stops to express, and production efficiency is very low.In order to overcome batch these drawbacks of cultivating, developed feeding culture (Fed-batch), promptly in culturing process,, in reactor, add a certain or several specific restricted substrates continuously or off and on according to the cellular metabolism demand.Feeding culture is compared with batch cultivation, culture cycle prolongs, can finally obtain higher viable cell density and production concentration (J.B.Griffith, Animal Cell Culture and Production ofBiologicals, pp.401-410, Klumer Academic Publishers, R.Sasaki and K.Ikura (eds), (1991), Robbinson D K, Seamans T C, et al.Optimization of a fed-batch process for production ofa recombinant antibody.Annals NY Academy Sciences, (1994), 745:185-296.)

Representational example as,

(Xie LZ and Wang D I C.High cell density and high monoclonal antibodyproduction through medium design and rational control in a bioreactor.Biotechnol.Bioeng such as Xie, (1996), 51:725-729.) form (protein according to cell, RNA, DNA, fat, carbohydrate) and product form, VITAMIN and ATP demand design nutrient concentrations and stream rate of acceleration, when glucose and glutamine during at lower concentration, according to cell density and growth velocity decision stream rate of acceleration.Cell density and monoclonal antibody concentration have improved 2 times and 10 times respectively in this way, and monoclonal antibody concentration reaches 0.5g/L in the square vase, reach 0.9g/L in the reactor.

(Zhou WC, Rehm J ﹠amp such as Zhou; Hu WS.High viable cell concentration fed-batch culturesof Hybridoma through on-line nutrient feeding.Biotechnol Bioeng, (1995), 46:579-587.) by online detection OUR, and by the stoichiometric relation between cell oxygen consumption and the nutrient consumption, dynamic flow adds salt-free complete enrichment medium and carries out the hybridoma feeding culture, and viable cell density has reached 1.2 * 10 ⁷Cells/mL, cellular metabolism is converted into the low generation of lactic acid and ammonia, the high flow rate of oxygen from the height generation of lactic acid and ammonia, the low consumption of oxygen.

But the stream that will reach optimization adds process, and key is the optimal control of cell in the implementation procedure, the controlled target of promptly studying the metabolic rule of cell and determining to optimize.Obviously, though the method for Xie etc. seems accurately, too complexity must cause research cycle long and workload is huge.Simultaneously, in zooblast feeding culture process, the metabolic rule of cell and physiological status are not invariable, therefore be feedback and the correction that does not all have concrete consideration process in the design of Xie or the method for Zhou, in case there is the instability that just will cause process in difference, even disperse, cause final failure.

Summary of the invention

The technical issues that need to address of the present invention are to disclose a kind of animal cell feeding culture process, to satisfy the needs of relevant field development.

Method of the present invention comprises the steps:

(a). be used for the feeding culture process of metabolic rule research.Obtain the metabolic rule of cell under different physiological statuss, set up the metabolic kinetic model of cell; Determine the optimal control target of feeding culture; Determine the fed-batch medium component proportions of optimization according to the dynamic metabolism of cell.

(b). be used for the optimization feeding culture process of high-density culture.According to the operating parameters of equipment with the optimization fed-batch medium component proportions that obtains (a), determine that the substratum of optimizing forms; Exponential growth rule according to cell is carried out the exponential feeding culture, and the regulating cell physiological status is in the controlled target of optimization; Simultaneously, the concentration of ammonia in cell density that obtains according to taking a sample and the culture environment, feedback modifiers stream rate of acceleration is avoided dispersing of system.

Route in the enforcement,

(a). metabolic rule and definite optimization aim of research cell

Studying cell and be in metabolic rule under the different physiological statuss, is to adopt batch culture assays or so-called pulse testing method traditionally.In batch culturing process between sampling nutrient concentrations alter a great deal, present serious time variation and uncertainty, uncontrollable culture environment and state bring very big difficulty especially for the growth metabolism rule of research cell under low nutrient concentrations.The pulse testing method seems rationally, but cell has the process of an adaptation to the variation of culture environment, is in suddenly under a kind of new environment and can not presents real metabolic rule at once.

Can address this problem on the method for the present invention,, can realize the long-term quasi-stable state growth of cell under different culture condition by rational design fed-batch medium and stream rate of acceleration.Simultaneously, because continuous perseveranceization cultivation of feeding culture period ratio or perfusion culture will be lacked a lot, just, provide convenience for obtaining the metabolic rule of cell under different physiological statuss apace and determining the optimal control target.

Because it is very complicated that substratum is formed, and often exist between various composition substitute, interaction such as complementation, the global optimization design is very complicated.Simplified process method is, investigates the metabolic rule under several extreme cultivation conditions, dwindles the optimizing scope and progressively determines optimization aim.Because lactic acid and ammonia are the main by products that restrictive cell density and production concentration improve, general strategy be restriction in the culture environment glucose or (with) glutamine concentration, therefore, the metabolic rule of high spot reviews cell under glucose limitation and glutamine limited state.Be to realize corresponding state quasi-stable state growth down, design fed-batch medium and flowed rate of acceleration, reasonably suppose and estimate, design corresponding stream and add process according to the basic dynamic parameter according to batch experimental data of cultivating.

Feeding culture with glucose limitation is designed to example:

According to batch experimental result of cultivating, as Glucose＜0.5mmol/L, Glutamine＞0.3mmol/L, and other nutrition can be thought to be in the glucose limitation cultivation conditions when not being construed as limiting.Simultaneously, for getting rid of the influence of by product as far as possible, need Lactate＜40mmol/L, Ammonia＜6mmol/L under the above-mentioned cultivation conditions to growth.Tentatively obtain under this state by the former experiments result: q _Gluc1.3mmol/ (10 ⁹Cellsday), q _Gln0.65mmol/ (10 ⁹Cellsday), q _NH30.5mmol/ (10 ⁹Cellsday), Y _NH3/gln0.75.The bio-reactor of 2L is adopted in experiment, initial incubation volume 700ml, and the stream rate of acceleration is chosen as 200ml/day, can estimate that thus stream adds thinning ratio D0.3 in the process～0.1 1/day.Initial substratum is Hybri SFM I (this laboratory oneself exploitation), and cell density is greater than 1.0 * 10 ⁶Begin stream during mmol/L and add, this moment, the concentration of ammonia was about 2.0mmol/L, and the average specific generating rate of longer for some time inner cell was about 0.5 1/day after stream added.

The estimation of maximum viable cell density (at this moment, thinking it mainly is that by product ammonia suppresses to cause stopping of cell growth),

Design is when X3.0 * 10 ⁶Reach glucose limitation during cells/L, at this moment D0.25 1/day.

The estimation of glucose concn in the fed-batch medium,

$\mathrm{Gluc}=\frac{q_{s}X}{D}=\frac{1.3\×3}{0.25}=15.6\mathrm{mmol}/L,$ Get Gluc=16mmol/L.

The estimation of glutamine concentration in the fed-batch medium,

$G\ln \≥S+\frac{q_{s}X}{D}=0.3+\frac{0.65\×3}{0.25}=8.1\mathrm{mmol}/L$ (avoiding the substrate glutamine limited)

${G\ln }_{\mathrm{in}}\≤S+\frac{q_s{X}_{v\max }}{D}=S+\frac{q_p}{Y_{P/S}}\·\frac{X_{v\max }}{D}\≈S+\frac{P}{Y_{P/S}}=0.3+\frac{6}{0.75}=8.3\mathrm{mmol}/L$

(ammonia suppresses, $\frac{\mathrm{dP}}{\mathrm{dt}}\≈0$ ) _ Election Gln=8.3mmol/L Xia

Other nutrition such as amino acid concentration are determined with the proportionlity of glucose or glutamine specific consumption rate according to them.

Relatively the metabolic rule of cell under different states determined the training objective of optimizing.Simultaneously, obtain the dynamic metabolism relation of cell:

$\left[1\right].---q_s=\frac{1}{{\mathrm{VX}}_v}[{\mathrm{FS}}_{\mathrm{in}}-\frac{d\left(\mathrm{VS}\right)}{\mathrm{dt}}],q_p=\frac{1}{V{X}_v}\frac{d\left(\mathrm{VP}\right)}{\mathrm{dt}}$

[2].C _Gln＝f(q _NH3)

According to [1], determine the ratio of the various nutritive ingredients of substratum.

(b). the feeding culture process of optimization

Be in cell under the specific physiological status and be amplification growth exponentially, therefore being in stream under the targeted physiological condition of optimization with regulating cell, to add process also be the process that index stream adds.But in culturing process, the actual physiological status of cell still can constantly change, and therefore, in actually operating, preferably feedback modifiers stream adds process further.

Can only replace with the countless stream of constant speed stage by stage Calais on index stream is added in and implements, the stream rate of acceleration in each time period is determined the mean value of time by theoretical index stream rate of acceleration in this stage.At t _iTo t _I+1Between, quasi-stable state is handled and (to be supposed that cell is in specific physiological status, i.e. μ, q _sConstant), and hypothesis μ _iμ _I-1, $q_{s_i}\≈q_{s_{i-1}},$ The stream rate of acceleration is determined by following formula:

$\left[3\right].---F_i=\stackrel{\‾}{F\left(t_{i\→i+1}\right)}={\∫}_{t_i}^{t_{i+1}}F\left(t\right)\mathrm{dt}/(t_{i+1}-t_i)=\frac{b_i{X}_i{V}_i\left[\exp \right[{\mathrm{\μ}}_i(t_{i+1}-t_i)]-1]}{t_{i+1}-t_i}$

Wherein:

${\mathrm{\μ}}_i=\frac{d\ln X_i{V}_i}{\mathrm{dt}}\≈{\mathrm{\μ}}_{i-1}\≈\frac{\ln (X_i{V}_i/X_{i-1}{V}_{i-1})}{t_i-t_{i-1}}$

$b_i=\frac{1}{Y_{x/s_i}\·S_{\mathrm{in}}}=\frac{q_{s_i}}{S_{\mathrm{in}}{\mathrm{\μ}}_i}\≈\frac{q_{s_{i-1}}}{S_{\mathrm{in}}{\mathrm{\μ}}_{i-1}}$

$q_{s_{i-1}}=\frac{1}{{\left(\stackrel{\‾}{\mathrm{VX}}\right)}_i}{F}_{i-1}(S_{\mathrm{in}}-S_{i-1}){\≈F}_{i-1}{S}_{\mathrm{in}}\·[\ln \frac{V_i{X}_i}{V_{i-1}{X}_{i-1}}/(V_i{X}_i-V_{i-1}{X}_{i-1})]$

If constant duration (t _i-t _I-1=t _I+1-t _i) sampling, by the iterative relation (Feed-forward algorithm) that must stream with co-relation adds control be:

$\left[4\right].{---F}_i=\frac{X_i{V}_i}{X_{i-1}{V}_{i-1}}\·F_{i-1}$

Wherein,

V _i=V _I-1+ F _I-1(t _i-t _I-1)-(v _{The Election sample}) _i

Consider that the physiological status that stream adds cell reality in the process constantly changes, experimental stage is dwindled the sampling interval as far as possible and is detected the concentration of ammonia, according to the metabolic kinetic model feedback modifiers stream rate of acceleration that obtains in [2].

More specifically method comprises the steps:

Is 1.5～2.5 * 10 with cell with viable cell density ⁵Cells/ml is seeded in the bio-reactor, when viable cell density reaches 0.8～1.2 * 10 ⁶Behind the cells/ml, the beginning supplemented medium, thinning ratio is 0.2～0.3day ^-1

Said substratum is glucose limitation fed-batch medium or glutamine limited fed-batch medium, and glucose and glutamine concentration are respectively 7.8～17.5mmol/L and 6.0～10.0mmol/L in the glucose limitation fed-batch medium; Glucose and glutamine concentration are respectively 17.5～30mmol/L and 2.5～5.0mmol/L in the glutamine limited fed-batch medium, and other composition is as shown in the table:

Composition
		Inorganic salt (mg/L)
CaCl 2	116.60
		CuSO 4·5H 2O	0.0013
Fe(NO 3) 3·9H 2O	0.05
		FeSO4·7H 2O	0.417
KCl	311.80
		MgCl 2	28.64
MgSO 4	48.84
		NaCl	6995.50
aHCO 3	2440
		NaH 2PO 4H 2O	62.50
Na 2HPO 4	71.02
		ZnSO 4·7H 2O	0.432
		L-amino acid (mmol/L)
Alanine	0.050
		Arginine·HCl	0.700-1.000
Asparagine·H 2O	0.050-0.450
		Aspartic acid	0.050-0.200
Cysteine·H 2O	0.100-0.200
		Cystein·2HCl	0.100-0.200
Glutamic acid	0.050-0.200
		Glycine	0.250-0.500
Histidine·HCl·H 2O	0.150-0.500
		Isoleucine	0.400-1.000
Leucine	0.450-1.200
		Lysine·HCl	0.500-1.000
Methionine	0.110-0.400
		Phenylalanine	0.200-0.500

Proline	0.150-0.300
		Serine	0.250-0.900
Threonine	0.450-0.800
		Tryptophan	0.040-0.250
Tyrosine·2Na·2H 2O	0.200-0.500
		Valine	0.450-1.000
		Vitamins/cofactors(mg/L)
Biotin	0.0035
		Pantothenate·Ca	2.24
Choline·Cl	8.98
		Folic acid	2.65
Inositol	12.60
		Niacinamide	2.02
Pyridoxine·HCl	2.031
		Riboflavin	0.219
Thiamine·HCl	2.17
		Thymidine	0.365
Vitamin B 12	0.68
Trace element (nmol/L)
		H 2SeO 3	10
MnSO 4·4H 2O	1.0
		Na 2SiO 3·5H 2O	50
(NH 4) 6Mo 7O 24·4H 2O	1.0
		NH 4VO 3	5
NiCl 2·6H 2O	0.5
		SnCl 2·2H 2O	0.5
		Other (mg/L)
Na Hypoxanthine	2.39
		Linoleic acid	0.042
Lipoic acid	0.105
		Phenol red	8.10
Sodium Putrescine·2HCl	0.081
		Pyruvate·Na	220.00
Primaton RL	1000
		Regular Insulin	5-10
Transferrins,iron complexes	5-10
		Albumin	0-100
		Other (μ mol/L)
Hydrocortisone	0.1-1.0
		Dexamethasone	0.1-1.0
The B-mercaptoethanol	10-30
		Estradiol	0.01
Progesterone	0.02
		Thanomin	20-100

Cultural method employing document (Li Dongxiao, Zhang Shuxiang, Zhu Minglong, etc. glucose and glutamine concentration are to the influence of hybridoma growth, East China University of Science's journal, 2003,29 (4): 359-362.) reported method.Controlling reactor pH=7.2 ± 0.1, dissolved oxygen (DO) is 50% air saturation, temperature is 36.8, mixing speed 120rpm.

The glucose limitation feeding culture reached the quasi-stable state of glucose limitation between 110～140 hours, the glutamine limited feeding culture reached the quasi-stable state of glutamine limited between 96～144 hours.

(b). the feeding culture process of optimization

Is 1.5～2.5 * 10 with cell with viable cell density ⁵Cells/ml is seeded in the bio-reactor, and the concentration of glucose and glutamine is respectively 7.8mmol/L and 2.5mmol/L in the initial substratum.When viable cell density reaches 0.8～1.2 * 10 ⁶Behind the cells/ml, the beginning supplemented medium.Other conditions are the same.

The feeding culture based component of optimizing is as shown in the table:

Composition
		Inorganic salt (mg/L)
CaCl 2	116.60
		CuSO 4·5H 2O	0.0013
Fe(NO 3) 3·9H 2O	0.05
		FeSO4·7H 2O	0.417
KCl	311.80
		MgCl 2	28.64
MgSO 4	48.84
		NaCl	6995.50
NaHCO 3	2440
		NaH 2PO 4H 2O	62.50
Na 2HPO 4	71.02
		ZnSO 4·7H 2O	0.432
		L-amino acid (mmol/L)
Alanine	0.050
		Arginine·HCl	1.380-5.520
Asparagine·H 2O	0.730-2.940
		Aspartic acid	0.090-0.400
Cysteine·H 2O	0.170-0.500
		Cystein·2HCl	0.400-1.600
Glutamic acid	0.400-1.600
		Glutamine	16.500-66.000
Glycine	0.500
		Histidine·HCl·H 2O	0.500-2.000
Isoleucine	1.800-7.200
		Leucine	1.900-7.600
Lysine·HCl	1.200-4.800
		Methionine	0.3800-1.300.
Phenylalanine	0.750-3.000
		Proline	0.400-1.600
Serine	0.400-1.600
		Threonine	1.100-4.400
Tryptophan	0.250-1.000
		Tyrosine·2Na·2H 2O	1.150-4.600
Valine	1.400-5.600
Vitamins/cofactors(mg/L)
		Biotin	0.0035
Pantothenate·Ca	2.24

Choline·Cl	8.98
		Folic acid	2.65
Inositol	12.60
		Niacinamide	2.02
Pyridoxine·HCl	2.031
		Riboflavin	0.219
Thiamine·HCl	2.17
		Thymidine	0.365
Vitamin B 12	0.68
Trace element (nmol/L)
		H 2SeO 3	10
MnSO 4·4H 2O	1.0
		Na 2SiO 3·5H 2O	50
(NH 4) 6Mo 7O 24·4H 2O	1.0
		NH 4VO 3	5
NiCl 2·6H 2O	0.5
		SnCl 2·2H 2O	0.5
		Other composition (mg/L)
Glucose	9000-36000
		Na Hypoxanthine	2.39
Linoleic acid	0.042
		Lipoic acid	0.105
Phenol red	8.10
		Sodium Putrescine·2HCl	0.081
Pyruvate·Na	220.00
		Primaton RL	1000
Regular Insulin	5
		Transferrins,iron complexes	10
Albumin	50-100
Other composition (μ mol/L)
		Hydrocortisone	0.3-2.0
Dexamethasone	0.3-2.0
		The B-mercaptoethanol	10
Estradiol	0.01
		Progesterone	0.02
Thanomin	200-500

Method of the present invention has the following advantages:

1. be widely used in the cultivation of a plurality of cell strains or clone;

2. can reach the quasi-stable state of cell under varying environment apace and cultivate, be convenient to the research of cellular metabolism rule and the foundation of metabolic kinetic model;

3. set up the index feeding culture model of optimizing, can realize with regulation and control zooblast physiological status being that the optimization stream of direct target adds process;

4. consider the feedback and the correction of Controlling System, avoided dispersing of process;

5. easy and simple to handle, be suitable for instructing large-scale industrial production.

Description of drawings

Fig. 1 is the optimization feeding culture control flow chart of band feedback.

Fig. 2 is the cell growth figure of the feeding culture under the HB58 hybridoma glucose limitation.

Fig. 3 is the feeding culture cellular metabolism figure under the HB58 hybridoma glucose limitation.

Fig. 4 is the feeding culture cell growth figure under the HB58 hybridoma glutamine limited.

Fig. 5 is the feeding culture cellular metabolism figure under the HB58 hybridoma glutamine limited.

Fig. 6 optimizes the growth of feeding culture process cell for the HB58 hybridoma and antibody generates figure.

Embodiment

Referring to Fig. 1, initial flow rate of acceleration F ₀According to the add some points specific growth rate (μ of cell of stream ₀), viable cell density (X ₀), volume of culture (V ₀), the concentration (S of manipulated variable glutamine ₀), the content (S of glutamine in the fed-batch medium _In) and sampling interval (Δ t) equivalent determine.General stream adds only needs uniformly-spaced time sampling, counting cells density (X in the process _v), can determine stream rate of acceleration next time.In addition, in order further to improve the stability of whole process, measure ammonia concentration (C during sampling simultaneously _NH3), calculate the specific production rate (q of ammonia at this moment _NH3), according to the metabolic kinetic model (C of this cell _{GlnBioreactor}=f (q _NH3)) can determine the concentration (C of glutamine in culture environment this moment _{GlnBioreactor}).Controlling concn (C according to the manipulated variable glutamine _GlnSetpoint), further obtain the stream rate of acceleration (F that revises _IModified).

This cell strain (being) that obtains according to experimental stage adds related law in the process optimizing stream, further instructs the actual production stage, avoids the too frequent interval sampling of practical stage.

Nomenclature:

The defined declaration of above-mentioned each symbol is as follows:

Gluc---glucose

Gln---glutamine

The concentration of C---substrate or product

D---release rate

F---stream rate of acceleration (Feeding rate, ml/day)

P---product

Specific consumption (generation) speed of q---substrate (product)

S _In---the substrate in the fed-batch medium

T---the time

V---reactor volume

X _v---viable cell (Viable cells, 10 ⁶Cells/ml)

Y _X/s---born of the same parents are to the productive rate of substrate

The specific growth rate of μ---cell

Embodiment 1～2

(a). metabolic rule research

With method of the present invention the physiological status of HB58 hybridoma (ATCC) under glucose limitation or glutamine limited culture condition studied.In 2 liters of bio-reactors of Biostat B (German B.BRAUN company), inoculate inoculum density 2.0 * 10 ⁵Cells/ml.When viable cell 0 density reaches 1.0 * 10 ⁶Begin feeding culture behind the cells/ml, thinning ratio is 0.30day ^-1, incubation time was respectively 125 hours and 100 hours, had realized that the quasi-stable state of glucose limitation (seeing Fig. 2 and Fig. 3) and glutamine limited is cultivated (Fig. 4 and Fig. 5), can be used for studying when cell being in the corresponding state metabolic rule in following time.The reactor mixing speed is 120rpm in the whole culturing process, and temperature is 36.8 ℃, and dissolved oxygen is 50% air saturation.

The component and the concentration of glucose limitation fed-batch medium are as follows:

Composition
		Inorganic salt (mg/L)
CaCl 2	116.60
		CuSO 4·5H 2O	0.0013
Fe(NO 3) 3·9H 2O	0.05
		FeSO4·7H 2O	0.417
KCl	311.80
		MgCl 2	28.64
MgSO 4	48.84
		NaCl	6995.50
NaHCO 3	2440
		NaH 2PO 4H 2O	62.50
Na 2HPO 4	71.02
		ZnSO 4·7H 2O	0.432
		L-amino acid (mmol/L)
Alanine	0.050
		Arginine·HCl	0.700
Asparagine·H 2O	0.100
		Aspartic acid	0.100
Cysteine·H 2O	0.100
		Cystein·2HCl	0.200
Glutamic acid	0.100
		Glutamine	8.500
Glycine	0.250
		Histidine·HCl·H 2O	0.250
Isoleucine	0.600
		Leucine	0.650
Lysine·HCl	0.500
		Methionine	0.200
Phenylalanine	0.350
		Proline	0.150
Serine	0.500
		Threonine	0.650
Tryptophan	0.150
		Tyrosine·2Na·2H 2O	0.200

Valine	0.800
Vitamins/cofactors(mg/L)
		Biotin	0.0035
Pantothenate·Ca	2.24
		Choline·Cl	8.98
Folic acid	2.65
		Inositol	12.60
Niacinamide	2.02
		Pyridoxine·HCl	2.031
Riboflavin	0.219
		Thiamine·HCl	2.17
Thymidine	0.365
		Vitamin B 12	0.68
		Trace element (nmol/L)
H 2SeO 3	10
		MnSO 4·4H 2O	1.0
Na 2SiO 3·5H 2O	50
		(NH 4) 6Mo 7O 24·4H 2O	1.0
NH 4VO 3	5
		NiCl 2·6H 2O	0.5
SnCl 2·2H 2O	0.5
Other composition (mg/L)
		Glucose	2900
Na Hypoxanthine	2.39
		Linoleic acid	0.042
Lipoic acid	0.105
		Phenol red	8.10
Sodium Putrescine·2HCl	0.081
		Pyruvate·Na	220.00
Primaton RL	1000
		Regular Insulin	10
Transferrins,iron complexes	10
		Albumin	100
		Other (μ mol/L)
Hydrocortisone	0.2
		Dexamethasone	0.2
The B-mercaptoethanol	10
		Estradiol	0.01
Progesterone	0.02
		Thanomin	100

The component and the concentration of glutamine limited fed-batch medium are as follows:

Composition
		Inorganic salt (mg/L)
CaCl 2	116.60
		CuSO 4·5H 2O	0.0013
Fe(NO 3) 3·9H 2O	0.05
		FeSO4·7H 2O	0.417
KCl	311.80

MgCl 2	28.64
		MgSO 4	48.84
NaCl	6995.50
		NaHCO 3	2440
NaH 2PO 4H 2O	62.50
		Na 2HPO 4	71.02
ZnSO 4·7H 2O	0.432
L-amino acid (mmol/L)
		Alanine	0.050
Arginine·HCl	0.900
		Asparagine·H 2O	0.450
Aspartic acid	0.200
		Cysteine·H 2O	0.200
Cystein·2HCl	0.200
		Glutamic acid	0.200
Glutamine	2.500
		Glycine	0.250
Histidine·HCl·H 2O	0.350
		Isoleucine	0.600
Leucine	0.800
		Lysine·HCl	0.800
Methionine	0.250
		Phenylalanine	0.400
Proline	0.150
		Serine	0.450
Threonine	0.650
		Tryptophan	0.090
Tyrosine·2Na·2H 2O	0.400
		Valine	0.900
		Vitamins/cofactors(mg/L)
Biotin	0.0035
		Pantothenate·Ca	2.24
Choline·Cl	8.98
		Folic acid	2.65
Inositol	12.60
		Niacinamide	2.02
Pyridoxine·HCl	2.031
		Riboflavin	0.219
Thiamine·HCl	2.17
		Thymidine	0.365
Vitamin B 12	0.68
Trace element (nmol/L)
		H 2SeO 3	10
MnSO 4·4H 2O	1.0
		Na 2SiO 3·5H 2O	50
(NH 4) 6Mo 7O 24·4H 2O	1.0
		NH 4VO 3	5
NiCl 2·6H 2O	0.5
		SnCl 2·2H 2O	0.5
		Other composition (mg/L)
Glucose	5000

Na Hypoxanthine	2.39
		Linoleic acid	0.042
Lipoic acid	0.105
		Phenol red	8.10
Sodium Putrescine·2HCl	0.081
		Pyruvate·Na	220.00
Primaton RL	1000
		Regular Insulin	10
Transferrins,iron complexes	10
		Albumin	100
		Other composition (μ mol/L)
Hydrocortisone	0.2
		Dexamethasone	0.2
The B-mercaptoethanol	10
		Estradiol	0.01
Progesterone	0.02
		Thanomin	100

Among Fig. 2, curve 1 is represented viable cell, and curve 2 is represented dead cell, and curve 3 is represented specific growth rate, and curve 4 is represented thinning ratio;

Among Fig. 3, curve 5 is represented glucose, and curve 6 is represented lactic acid, and curve 7 is represented L-Ala, and curve 8 is represented glutamine, and curve 9 is represented ammonia;

Among Fig. 4, curve 10 is represented viable cell, and curve 11 is represented dead cell, and curve 12 is represented specific growth rate, and curve 13 is represented thinning ratio;

Among Fig. 5, curve 14 is represented glucose, and curve 15 is represented lactic acid, and curve 16 is represented L-Ala, and curve 17 is represented glutamine, and curve 18 is represented ammonia;

Embodiment 3～4

(b). high-density is optimized feeding culture

(ATCC) is optimized feeding culture with method of the present invention with the HB58 hybridoma.In 2 liters of bio-reactors of Biostat B (German B.BRAUN company), the viable cell inoculum density is 2.0 * 10 ⁵Cells/ml.Glucose and glutamine concentration are respectively 7.8mmol/L and 2.5mmol/L in the initial substratum, and other amino acid concentration is the original concentration of DMEM/F12 (1: 1) substratum.When viable cell density reaches 1.0 * 10 ⁶Behind the cells/ml, beginning is according to cell requirements supplemented medium exponentially.It is 0.30day that stream adds thinning ratio ^-1

The concentration of glucose and glutamine is respectively 100mmol/L and 33mmol/L in the fed-batch medium, and amino acid is 6.5 times of DMEM/F12 (1: 1, mass ratio) substratum quality.The reactor mixing speed is 120rpm in the whole culturing process, and temperature is 36.8 ℃, and dissolved oxygen is 50% air saturation.

Concrete component and the concentration of the fed-batch medium of optimizing is as follows:

Composition
		Inorganic salt (mg/L)
CaCl 2	116.60
		CuSO 4·5H 2O	0.0013
Fe(NO 3) 3·9H 2O	0.05
		FeSO4·7H 2O	0.417
KCl	311.80
		MgCl 2	28.64
MgSO 4	48.84
		NaCl	6995.50
NaHCO 3	2440
		NaH 2PO 4H 2O	62.50
Na 2HPO 4	71.02
		ZnSO 4·7H 2O	0.432
		L-amino acid (mmol/L)
Alanine	0.050
		Arginine·HCl	2.760
Asparagine·H 2O	1.470
		Aspartic acid	0.170
Cysteine·H 2O	0.330
		Cystein·2HCl	0.650
Glutamic acid	0.170
		Glutamine	33.000
Glycine	0.500
		Histidine·HCl·H 2O	0.980
Isoleucine	3.600
		Leucine	3.800
Lysine·HCl	2.250
		Methionine	0.650
Phenylalanine	1.500
		Proline	0.820
Serine	0.800
		Threonine	2.200
Tryptophan	0.500
		Tyrosine·2Na·2H 2O	2.140
Valine	2.800
Vitamins/cofactors(mg/L)
		Biotin	0.0035
Pantothenate·Ca	2.24
		Choline·Cl	8.98
Folic acid	2.65
		Inositol	12.60
Niacinamide	2.02
		Pyridoxine·HCl	2.031
Riboflavin	0.219
		Thiamine·HCl	2.17
Thymidine	0.365
		Vitamin B 12	0.68

Trace element (nmol/L)
		H 2SeO 3	10
MnSO 4·4H 2O	1.0
		Na 2SiO 3·5H 2O	50
(NH 4) 6Mo 7O 24·4H 2O	1.0
		NH 4VO 3	5
NiCl 2·6H 2O	0.5
		SnCl 2·2H 2O	0.5
		Other composition (mg/L)
Glucose	18000
		Na Hypoxanthine	2.39
Linoleic acid	0.042
		Lipoic acid	0.105
Phenol red	8.10
		Sodium Putrescine·2HCl	0.081
Pyruvate·Na	220.00
		Primaton RL	1000
Regular Insulin	5
		Transferrins,iron complexes	10
Albumin	50
Other composition (μ mol/L)
		Hydrocortisone	0.2
Dexamethasone	0.2
		The B-mercaptoethanol	10
Estradiol	0.01
		Progesterone	0.02
Thanomin	300

Maximum viable cell density reaches 5.66 * 10 in the feeding culture process ⁶Cells/ml, monoclonal antibody concentration reaches the 379mg/L (see figure 6), and the result who criticizes cultivation with ordinary culture medium compares, and has improved 2.9 times and 3.1 times respectively.In culturing process, glucose and glutamine stable maintenance are in lower concentration, and wherein glucose concn is below 0.5mmol/L, and glutamine concentration remains between the 0.1-0.2mmol/L, metabolic by-prods has obtained control preferably, and ammonia concentration is up to 4.4mmol/L.

Among Fig. 6, curve 19 is represented viable cell, the total cell of curve 20 representatives, and curve 3 is represented antibody.

Claims (5)

1. the feeding culture process of a system is used for the metabolic rule research of zooblast and optimizes suspension culture, it is characterized in that, comprises the steps:

(a). add process according to batch culturing cell growth metabolism dynamics Design initial medium, fed-batch medium and stream, realize different quasi-stable state culture environment, the metabolic rule of research cell under different states determined the controlled target of optimizing; Set up the metabolic kinetic model of cell; Determine the fed-batch medium component proportions of optimization according to the dynamic metabolism of cell;

(b). according to the operating parameters of equipment with the medium component ratio of the optimization that obtains (a), determine that the substratum of optimizing forms.Stream adds the controlled target that regulating cell physiological status in the process is in optimization, simultaneously, the concentration of ammonia in cell density that obtains according to sampling and the culture environment, feedback modifiers stream rate of acceleration is avoided dispersing of system.

2. method according to claim 1 is characterized in that, step (a) comprises the steps:

Is 1.5～2.5 * 10 with cell with viable cell density ⁵Cells/ml is seeded in the bio-reactor, when viable cell density reaches 0.8～1.2 * 10 ⁶Behind the cells/ml, the beginning supplemented medium, thinning ratio is 0.2～0.3day ^-1

Said substratum is glucose limitation fed-batch medium or glutamine limited fed-batch medium; Glucose and glutamine concentration are respectively 7.8～17.5mmol/L and 6.0～10.0mmol/L in the glucose limitation fed-batch medium; Glucose and glutamine concentration are respectively 17.5～30mmol/L and 2.5～5.0mmol/L in the glutamine limited fed-batch medium, and other composition is as shown in the table: Composition Inorganic salt (mg/L) CaCl ₂ 116.60 CuSO ₄·5H ₂O 0.0013 Fe(NO ₃) ₃·9H ₂O 0.05 FeSO ₄·7H ₂O 0.417 KCl 311.80 MgCl ₂ 28.64 MgSO ₄ 48.84 NaCl 6995.50 aHCO ₃ 2440 NaH ₂PO ₄H ₂O 62.50 Na ₂HPO ₄ 71.02 ZnSO ₄·7H ₂O 0.432 L-amino acid (mmol/L) Alanine 0.050 Arginine·HCl 0.700-1.000 Asparagine·H ₂O 0.050-0.450 Aspartic acid 0.050-0.200 Cysteine·H ₂O 0.100-0.200 Cystein·2HCl 0.100-0.200 Glutamic acid 0.050-0.200 Glycine 0.250-0.500 Histidine·HCl·H ₂O 0.150-0.500 Isoleucine 0.400-1.000 Leucine 0.450-1.200 Lysine·HCl 0.500-1.000 Methionine 0.110-0.400 Phenylalanine 0.200-0.500 Proline 0.150-0.300 Serine 0.250-0.900 Threonine 0.450-0.800 Tryptophan 0.040-0.250 Tyrosine·2Na·2H ₂O 0.200-0.500 Valine 0.450-1.000 Vitamins/cofactors(mg/L) Biotin 0.0035 Pantothenate·Ca 2.24 Choline·Cl 8.98 Folic acid 2.65 Inositol 12.60 Niacinamide 2.02 Pyridoxine·HCl 2.031 Riboflavin 0.219 Thiamine·HCl 2.17 Thymidine 0.365 Vitamin B ₁₂ 0.68 Trace element (nmol/L) H ₂SeO ₃ 10 MnSO ₄·4H ₂O 1.0 Na ₂SiO ₃·5H ₂O 50 (NH ₄) ₆Mo ₇O ₂₄·4H ₂O 1.0 NH ₄VO ₃ 5 NiCl ₂·6H ₂O 0.5 SnCl ₂·2H ₂O 0.5 Other (mg/L) Na Hypoxanthine 2.39 Linoleic acid 0.042 Lipoic acid 0.105 Phenol red 8.10 Sodium Putrescine·2HCl 0.081 Pyruvate·Na 220.00 Primaton RL 1000 Regular Insulin 5-10 Transferrins,iron complexes 5-10 Albumin 0-100 Other (μ mol/L) Hydrocortisone 0.1-1.0 Dexamethasone 0.1-1.0 B-dredges basic ethanol 10-30 Estradiol 0.01 Progesterone 0.02 Thanomin 20-100

The glucose limitation feeding culture reached the quasi-stable state of glucose limitation between 110～140 hours, the glutamine limited feeding culture reached the quasi-stable state of glutamine limited between 96～144 hours.

3. method according to claim 1 is characterized in that, step (b) comprises the steps:

Is 1.5～2.5 * 10 with cell with viable cell density ⁵Cells/ml is seeded in the bio-reactor, and the concentration of glucose and glutamine is respectively 7.8mmol/L and 2.5mmol/L in the initial substratum, when viable cell density reaches 0.8～1.2 * 10 ⁶Behind the cells/ml, begin to add the fed-batch medium of optimization;

The feeding culture based component of optimizing is as shown in the table: Composition Inorganic salt (mg/L) CaCl ₂ 116.60 CuSO ₄·5H ₂O 0.0013 Fe(NO ₃) ₃·9H ₂O 0.05 FeSO4·7H ₂O 0.417 KCl 311.80 MgCl ₂ 28.64 MgSO ₄ 48.84 NaCl 6995.50 NaHCO ₃ 2440 NaH ₂PO ₄H ₂O 62.50 Na ₂HPO ₄ 71.02 ZnSO ₄·7H ₂O 0.432 L-amino acid (mmol/L) Alanine 0.050 Arginine·HCl 1.380-5.520 Asparagine·H ₂O 0.730-2.940 Aspartic acid 0.090-0.400 Cysteine·H ₂O 0.170-0.500 Cystein·2HCl 0.400-1.600 Glutamic acid 0.400-1.600 Glutamine 16.500-66.000 Glycine 0.500 Histidine·HCl·H ₂O 0.500-2.000 Isoleucine 1.800-7.200 Leucine 1.900-7.600 Lysine·HCl 1.200-4.800 Methionine 0.3800-1.300. Phenylalanine 0.750-3.000 Proline 0.400-1.600 Serine 0.400-1.600 Threonine 1.100-4.400 Tryptophan 0.250-1.000 Tyrosine·2Na·2H ₂O 1.150-4.600 Valine 1.400-5.600 Vitamins/cofactors(mg/L) Biotin 0.0035 Pantothenate·Ca 2.24 Choline·Cl 8.98 Folic acid 2.65 Inositol 12.60 Niacinamide 2.02 Pyridoxine·HCl 2.031 Riboflavin 0.219 Thiamine·HCl 2.17 Thymidine 0.365 Vitamin B ₁₂ 0.68 Trace element (nmol/L) H ₂SeO ₃ 10 MnSO ₄·4H ₂O 1.0 Na ₂SiO ₃·5H ₂O 50 (NH ₄) ₆Mo ₇O ₂₄·4H ₂O 1.0 NH ₄VO ₃ 5 NiCl ₂·6H ₂O 0.5 SnCl ₂·2H ₂O 0.5 Other composition (mg/L) Glucose 9000-36000 Na Hypoxanthine 2.39 Linoleic acid 0.042 Lipoic acid 0.105 Phenol red 8.10 Sodium Putrescine·2HCl 0.081 Pyruvate·Na 220.00 Primaton RL 1000 Regular Insulin 5 Transferrins,iron complexes 10 Albumin 50-100 Other composition (μ mol/L) Hydrocortisone 0-3-2.0 Dexamethasone 0.3-2.0 The B-mercaptoethanol 10 Estradiol 0.01 Progesterone 0.02 Thanomin 200-500

4. method according to claim 2 is characterized in that, said zooblast comprises hybridoma, CHO, NS0, BHK or 293 cells.

5. method according to claim 3 is characterized in that the substratum that is used is serum free medium.

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