CN107904200A - A kind of Combined culture base for expressing adalimumab and its application - Google Patents
A kind of Combined culture base for expressing adalimumab and its application Download PDFInfo
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- 239000002609 medium Substances 0.000 claims abstract description 85
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- 102220594896 Vasopressin-neurophysin 2-copeptin_M20A_mutation Human genes 0.000 claims abstract description 54
- 238000004113 cell culture Methods 0.000 claims abstract description 39
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- 239000008103 glucose Substances 0.000 claims description 77
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- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/241—Tumor Necrosis Factors
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- General Health & Medical Sciences (AREA)
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of Combined culture base for expressing adalimumab, it is made of basal medium and supplemented medium, wherein basal medium is by CD FortiCHO:M20A=1:0.5 1.5 compositions；Supplemented medium is by F001S:F001B=5 15:1 composition；Culture medium used in above-mentioned is commercial culture medium, a kind of any of the above described culture medium is used alone, the adalimumab of high yield, stabilization can not all be obtained, but it, which combines culture, can significantly improve the expression quantity of adalimumab, improve cell density and Cell viability, it can stablize and solve lactic acid in current Ah Da wood cell cultivation process and largely accumulate, the problem of destination protein low output, the yield and quality for being conducive to adalimumab further improves.
Description
Technical field
The present invention relates to a kind of Combined culture base for expressing adalimumab and its application, belongs to biotechnology and fermentation is led
Domain.
Background technology
Chinese hamster ovary (Chinese hamster ovary, CHO) cell derived is in the ovary of Chinese hamster.It is common
In biology and medical research, and the production for the treatment of recombinant protein.Relative to other cell expression systems, Chinese hamster ovary celI table
It can stably integrate with foreign gene up to system and be given expression to and native protein structure, immune by complicated posttranslational modification
The almost identical albumen such as originality, type of glycosylation and mode, so as to ensure the pharmacological property of recombinant protein and drug effect.In addition,
Chinese hamster ovary celI seldom secretes the intrinsic protein of itself, and the later stage for being also beneficial to foreign protein isolates and purifies.Therefore, Chinese hamster ovary celI at present
It has been the industrial production most common cell line of recombinant protein.
Recombinant monoclonal antibodies drug specificity is high and evident in efficacy, is one of hot spot medicine of Recent study, swollen
Before Clinics and Practices, autoimmune disease, infectious diseases and graft-rejection of knurl etc. have wide application
Scape.The characteristics of antibody drug is treated is that required dosage is big and treatment cycle is grown, therefore market demand is huge.Adalimumab
(Adalimumab) it is that a kind of human tumour necrosis factor (TNF-α) specificity that Abbott Laboratories develops recombinates IgG monoclonals
Antibody, is successively used for 8 kinds of rheumatoid arthritis, Crohn disease and psoriatic arthritis etc. certainly by USA and EU approval in recent years
The treatment of body immunity disease.Because of adalimumab good effect, Small side effects, clinically receive more and more attention.
Research currently for adalimumab cell culture is less, and the production of adalimumab is mainly with fed-batch
Training method, using Chinese hamster ovary celI, CD FortiCHO culture mediums carry out, and for expression quantity in 2-3g/L or so, expression quantity is relatively low, and
The generation and accumulation of accessory substance lactic acid are had in cell cultivation process, there is certain toxic action to cell, influence cell
Growth and product expression.
Patent document CN104212769 A provide a kind of cultural method in laboratory, and yield is 5.33g/L (and mesh
It is preceding highest in the prior art), but this is on the basis of collective media, and the additive of up to 16 kinds of addition, such as using should
Method carries out mass cell culture, and error is larger in operation, can not ensure the stability between batch.It is closest at present
The prior art be 104004814 A of CN (being directed to monoclonal antibody), destination protein content is relatively low.
Therefore, in the wooden field of cell culture of Ah Da, there is an urgent need to a kind of culture medium, it is desirable to the preparation component of the culture medium and
Method is simple, and same yield will also raise, and is further adapted to the requirement of the big production of cell culture, between guarantee production batch
Stability.
The content of the invention
In order to solve the above technical problem, the present invention provides a kind of Combined culture base for expressing adalimumab, by base
Basal culture medium and supplemented medium form, and the percentage by volume of basal medium and supplemented medium is respectively：55%-75% and
25%-45%；
Wherein basal medium is made of solution A and solution B, solution A:The volume ratio of B is 1：0.5-1.5, wherein solution A
Middle component is 20-28g/L CD FortiCHO, 0.9-1.2g/L poloxamer 188 and 4-8g/L glucose, remaining component
For water；Component is 20-28g/L M20A in solution B, remaining component is water；
Wherein supplemented medium is made of solution C and solution D, solution C:The volume ratio of D is 5-15：1, wherein in solution C
Component is 150-200g/L F001S, remaining component is water；Component is 50-100g/L F001B wherein in solution D, remaining component
For water.
The amount of preparation of wherein supplemented medium is according to each parameter index change in basal medium, carries out flexible modulation and matches somebody with somebody
System, determined including the concentration of glucose in basal medium, and cell growth condition, basal medium and feed supplement
The percentage by volume of culture medium is respectively：55%-75% and 25%-45%.
The purpose of solution C adjusts concentration of glucose and the supplement cell growth nutrition of basal medium.
The purpose of solution D is other nutrition needed for supplement cell growth.
Solution C and D must be added respectively under proper culture conditions can be only achieved optimal feed supplement effect, wherein C and D
Volume ratio be 5-15:In the case of 1, Cell viability reaches more than 80%.
In a preferred embodiment of the invention, the ratio of the basal medium and supplemented medium is among change
, the dosage increase of basal medium, the dosage of supplemented medium will be reduced accordingly.In the preferred embodiment of the present invention, with body
Product percentage calculates, and the volume of basal medium is 65%-75%, and the volume of the supplemented medium is 25%-35%.
The present invention does not have found close bibliography, CD FortiCHO are Chinese hamster ovary celIs in the screening of commercial medium
Most commonly seen culture medium is cultivated, and tri- kinds of culture mediums of remaining M20A, F001S and F001B are Shanghai De Siteli biotechnologys
The culture medium of Co., Ltd's production, these three culture mediums, disclosed information is less, does not also deliver pertinent literature.With the prior art
It is compared to the main distinction, the prior art generally uses a kind of commercial medium, or on a kind of basis of commercial base culture medium
Upper addition individual substance, such as copper ion, manganese ion, insulin, urea glycosides, there are no two kinds of commercial base culture mediums in proportion
Used after preparation.
CD FortiCHO are that chemical composition determines, without albumen, non-animal derived culture medium, for improving batch culture and mending
Expect the Chinese hamster ovary celI performance and yield of batch culture, the host cell proteins concentration in downstream purification process can be reduced, improve weight
The protein expression of growth and the suspension batch culture of group Chinese hamster ovary celI, suitable for CHO K1, GS CHO, CHO-STMCell line.
The chemically defined culture mediums of M20A are free of the component of any animal origin, its raw material composition has amino acid, vitamin, fat
Class etc., for improving batch the Chinese hamster ovary celI performance and yield of culture and fed-batch culture, improve recombinaant CHO cell growth and
The protein expression of suspension batch culture, suitable for CHO K1, GS CHO, CHO-STM cell lines.
F001S, F001B supplemented medium are free of the component of any animal origin, its raw material composition has amino acid, dimension life
Element, lipid, sugar etc., for improving the Chinese hamster ovary celI performance of fed-batch culture and yield, improve recombinaant CHO cell growth and
The protein expression of suspension batch culture, suitable for CHO K1, GS CHO, CHO-STM cell lines.
Report is used in combination seldom in commercial culture mediums different at present, and cause is that the component of commercial culture medium not
Bright, this is also combined the very big technical problem of band to commercial culture medium.
Existing technical problem is：It cannot simply be mixed between different culture mediums, prepare solution, often can be because of
Component therein is different or mixed proportion is different, occurs crystallizing after mixing or precipitates, causes nutritional ingredient smooth by cell
Absorb, cause cell mortality.
Therefore inventor has carried out multiple intersection Large-scale Screening for existing commercial culture medium, and screening process is shown in Table 1
With table 2：
The screening process of Combined culture base：Technical staff interacts work taking into full account between basal medium and supplemented medium
On the basis of, the method for taking comprehensive test, for the mixing ratio of different basal mediums, any two kinds of basal mediums
Example, different supplemented mediums, the mixed proportion of any two kinds of supplemented mediums, have carried out substantial amounts of screening test.
Found in the interpretation of result for carrying out the first round a large amount of screening tests, basal medium and ratio that following table is related to,
Supplemented medium and ratio can reach relatively good effect, carry out the second wheel screening on this basis, i.e., using examination comprehensively
The method tested, carries out 12 × 9=108 times experiment.It is shown in Table 1.
Table 1
According to second wheel screening test it turns out that, trained in the preferable experimental group of test effect using feed supplement
Support base F001S:F001B=10:1, accordingly, it is determined that joint supplemented medium and ratio, to further determine that commbined foundations are trained
Base and ratio are supported, technical staff has carried out third round screening test, as shown in table 2 below：
Table 2
It is described joint screening technique be related to culture basic variable it is very much, wherein many Medium Proportions are according to testing crew
Experience and knowledge determines in advance, therefore this joint screening technique is not that those of ordinary skill can subjective derive or root
According to the existing knowledge of commercial culture medium with regard to getable, substantial amounts of screening process can also be so saved.
According to the interpretation of result of third round screening test, from highest viable cell density, commbined foundations culture medium is equal
Higher than the highest viable cell density under the condition of culture of single basal medium, wherein, M20A:CD FortiCHO=1:3 cultures
Highest viable cell density highest under the conditions of base.Secondly M20A:CD FortiCHO=1:1 and M20A:CD FortiCHO=3:1
Under the conditions of, the highest viable cell density level under CD FortiCHO condition of culture is minimum.From Cell viability, thin
Cell viability in born of the same parents' incubation under five kinds of condition of culture is always maintained at well.From the point of view of destination protein content, from high to low
Condition of culture be M20A successively:CD FortiCHO=1:1, M20A:CD FortiCHO=1:3, M20A:CD FortiCHO
=3:1, M20A, CD FortiCHO.Therefore, consider every factor, determine M20A:CD FortiCHO=1:1 and M20A:
CD FortiCHO=1:3 are relatively suitable as the basal medium of technique amplification.
To further determine that the commbined foundations culture medium of large-scale production adalimumab, fourth round screening examination has been carried out
Test, i.e., carry out in the bioreactor.Table 3
Table 3
According to the interpretation of result of fourth round screening test, from the point of view of highest viable cell density, M20A:CD FortiCHO=
1:Highest viable cell density highest under the conditions of 1；From Cell viability, under the conditions of three kinds of different basal mediums, carefully
Born of the same parents' motility rate does not have notable difference；Analyzed from the change of lactic acid, when under three kinds of condition of culture by the 13rd day, except M20A:CD
FortiCHO=1:1 condition, the lactic acid concn in other two conditions has obvious ascendant trend, and such variation tendency may
Influence whether the motility rate of cell.From the point of view of destination protein content, M20A:CD FortiCHO=1:1 and 1:The 13rd day under the conditions of 3
Destination protein content reached 4.30g/L or so, it is higher than supreme good protein content in other conditions by about 20%.
The difference of several aspects such as comprehensive consideration highest viable cell density, Cell viability, lactic acid change, destination protein content
Judge, M20A:CD FortiCHO=1:1 as commbined foundations culture medium condition it is optimal, be ultimately determined to optimal joint culture
Base：Basal medium M20A:CD FortiCHO=1:1 and supplemented medium F001S:F001B=10:1.
The present invention furthermore provides a kind of Combined culture base, wherein, the body of the basal medium and supplemented medium
Product than being respectively for the percentage by volume of basic culture medium and supplemented medium：65%-75% and 25%-35%；
In this experimental program, the stream dosage of supplemented medium is simultaneously not fixed, but according in nutrient solution glucose it is dense
The growing state of degree and cell when the concentration of glucose in nutrient solution is less than 4-8g/L, especially 5g/L, then needs come what is determined
The amount for flowing the solution C added and D is calculated according to concentration of glucose and solution C and the volume ratio of D.In being preferable to carry out for the present invention
In example, the volume ratio preferred scope of basal medium and supplemented medium is 65%-75% and 25%-35%；
The preferable fed-batch mode of institute is in the embodiment of the present invention：During culture, daily according to concentration of glucose in nutrient solution,
The solution C and the amount of D that stream needed for calculating is daily adds, it is discontinuity disposably to add solution C and D, the fed-batch cultivation mode
Fed-batch cultivation.
The present invention furthermore provides a kind of Combined culture base, wherein, the solution A：The volume ratio of B is 1:1.
The present invention furthermore provides a kind of Combined culture base, wherein, component is 24.56g/L CD in the solution A
FortiCHO, 1g/L poloxamer 188 and 5g/L glucose, remaining component are water；Solution B is 27.62g/L M20A, its
Remaining component is water.
The present invention furthermore provides a kind of Combined culture base, wherein, the solution C：The volume ratio of D is 10：1.
Preferably, wherein component is the F001S of 164.9g/L in the solution C, remaining component is water；Wherein in solution D
Component is 76g/L F001B, remaining component is water.
The present invention furthermore provides a kind of preparation method of Combined culture base, comprises the steps of：
1) CD FortiCHO solid mediums are weighed, poloxamer188 and glucose are dissolved in the water, and are configured to solution
A；
2) M20A solid mediums are weighed, are dissolved in the water, are configured to solution B；
3) by solution A and B with 1:The volume ratio mixing of 0.5-1.5, obtains basal medium；
4) F001S and F001B are weighed, is dissolved in water, is configured to solution C and D, by solution C and D according to volume ratio
5-15:1 composition supplemented medium, solution C and D cannot be pre-mixed at this time；
5) basal medium has collectively constituted Combined culture base with supplemented medium described in step 4) in step 3).
Preferably, the method for the present invention is specially following steps：
1) CD FortiCHO solid mediums are weighed, poloxamer188 and glucose are dissolved in the water, and are configured to solution
A；
2) M20A solid mediums are weighed, are dissolved in the water, are configured to solution B；
3) by solution A and B with 1:1 volume ratio mixing, obtains basal medium；
4) F001S and F001B are weighed, is dissolved in water, is configured to solution C and D, by solution C and D according to volume ratio
10:1 is combined into supplemented medium, and solution C and D cannot be pre-mixed at this time；
5) by supplemented medium in basal medium in step 3) and step 4) according to percentage by volume 65%-75% and
The mode of 25%-35% is combined.
Two of which basal medium cannot carry out simple solid mixing, because after M20A need to first be dissolved in water, carry out
The detection of continuous item, after meeting all standard of M20A, can use；Such as various solid mediums are first mixed, afterwards plus water is matched somebody with somebody
System, then not only contain M20A but also FortiCHO containing CD, no examination criteria can carry out Quality Control.
Two kinds of supplemented mediums can not carry out simple solid mixing, and simply combine, i.e., described solution C and D cannot
Mixed, crystallization can be formed, it is necessary to which during stream adds, solution C and D are separately added into.
This is also an inventive point of preparation method of the present invention, and Combined culture is not mixed culture medium, it is necessary to from each other
Individualism, mutual association use, and particularly fluid infusion culture medium is also divided into two parts, and solution C and D need to be individually added into.
The present invention still further provides a kind of application of above-mentioned Combined culture base in adalimumab preparation, wherein,
The application comprises the steps of：
A) adalimumab cell line is inoculated with into the basal medium, Initial seeding density is 3-7 × 105cells/
ml；Cultivated at 36-38 DEG C of temperature 12-36 it is small when after, according to the concentration of glucose in basal medium, add feed-batch culture
Base, feed postition add for stream, and stream dosage ensures concentration of glucose and the stabilization of other nutrient levels；
B) when viable cell density reaches 12-18 × 106Cells/ml, 33-34 DEG C is down to by cultivation temperature；
C) other culture parameters are：PH 7.0 ± 0.2, dissolved oxygen 30-50.0%, initial speed 60-100rpm, top layer ventilation
It is 0.2-1lpm to measure as 0.2-1lpm, deep ventilation amount, after cultivating 10-15*24h, obtains the adalimumab cell culture
Liquid, by adalimumab purification step, obtains the adalimumab.
Wherein, the expression cell is mammalian cell, preferably Chinese hamster ovary celI.
Flow acceleration is 600~800ml/min
Preferably, the application comprises the steps of：
A) expression cell is inoculated with into the basal medium, Initial seeding density is 6 × 105cells/ml；In temperature
After when culture 24 is small at 36.7 DEG C, according to the concentration 5g/L of glucose in basal medium, supplemented medium, feed postition are added
Add for stream, flow acceleration is 600~800ml/min：Flow acceleration needs to ensure the stabilization of concentration of glucose；
B) when viable cell density reaches 15 × 106Cells/ml, 33.5 DEG C are down to by cultivation temperature；
C) other culture parameters are：PH 7.0 ± 0.2, dissolved oxygen 40.0%, initial speed 90rpm, top layer throughput are
0.5lpm, deep ventilation amount are 0.5lpm, after cultivating 12*24h, the adalimumab cell culture fluid are obtained, by Ah reaching
Wooden monoclonal antibody purification step, obtains the adalimumab.
The adalimumab purification step can refer to article " hydroxyapatite isolates and purifies anti-TNF alpha monoclonal antibody ".
LPM is the abbreviation (liter/min) of liter per minute
According to concentration of glucose in concentration of glucose addition control nutrient solution in 5g/L, progress Fed-Batch in step (a)
Culture.Glucose is important carbon source and the energy substance needed for Chinese hamster ovary celI growth metabolism, and energy is provided except being metabolized for cell growth
Outside, can generate accessory substance lactic acid under the catalytic action of lactic dehydrogenase, the accumulation of lactic acid can seriously affect cell growth and
The expression of product.When therefore supplying glucose with fed-batch mode in incubation and controlling it be in intermediate concentration, cell is given birth to
It is long to be significantly improved, and cell is improved significantly the utilization rate of glucose, so as to reduce the generation and accumulation of lactic acid.
During step (b) cooling culture, cultivation temperature is adjusted to 33.5 DEG C by 36.7 DEG C.Temperature is Chinese hamster ovary celI culture
Very important environmental parameter in journey, fast-growth is to plateau under the conditions of 36.7 DEG C for usual Chinese hamster ovary celI, when cell growth arrives
Up to design cell density when, i.e. (12-18) × 106Cells/ml, reduces cultivation temperature and cultivates rank to 33.5 DEG C into maintenance
Section.On the one hand cell metabolism can be reduced, higher Cell viability is maintained, strengthens the expression of adalimumab, is conducive to its quality
And structural homogeneity；Glucose has obtained more effective utilization under the conditions of another aspect Low- temperature culture, and then reduces accessory substance
The generation of lactic acid.
The purpose of the present invention：
1st, primary and foremost purpose of the invention be to overcome in terms of culture medium in the prior art there are the problem of, creative proposition
Two kinds of basal mediums and two kinds of supplemented mediums are used in combination, improve the yield of destination protein.
2nd, another object of the present invention is to overcome the prior art the shortcomings that and deficiency, in cell cultivation process, maintain
Suitable viable cell density and Cell viability, by the control to glucose and temperature, reduces the generation of accessory substance lactic acid
And accumulation, there is provided one kind is suitable for the CHO that large-scale production recombinates full people source anti-TNF alpha monoclonal antibody (adalimumab)
Cell culture processes and application.
The effect reached：
It is suitable for large-scale production the present invention relates to one kind and recombinates full people source anti-TNF alpha monoclonal antibody (adalimumab)
Chinese hamster ovary celI Combined culture base and its cultural method, higher viable cell density and cell can be kept in cell cultivation process
Motility rate, while the generation and accumulation of accessory substance lactic acid can be reduced, improve antibody production and quality.
(1) viable cell density：In cell cultivation process, if viable cell density is too low, destination protein yield is relatively low；If
Viable cell density is excessive, and nutrition supply deficiency, again results in the reduction of destination protein yield.Cell culture side provided by the invention
Method, in cell cultivation process, viable cell density can maintain of a relatively high level, and in 200L reactors, its highest is lived
Cell density is up to 39.66 × 106More than cells/ml, each nutriment is sufficient and is fully utilized, and adds destination protein
Yield.
(2) Cell viability：In cell cultivation process, the height of Cell viability is the important indicator for evaluating cell state,
When Cell viability is higher, the quality of the destination protein of generation is higher.But in rewinding, if Cell viability is excessive, one can be caused
Determine the waste of degree；Cell viability is too low, then can because increasing for impurity and caused by destination protein quality it is poor, and then increase downstream
Purification step.Cell culture processes provided by the invention, in 200L reactors, Cell viability is 85% or so during rewinding, both
It will not cause to waste, in turn ensure that the quality of destination protein, simplify downstream purification technological process.
(3) lactic acid content：In cell cultivation process, glucose for cell growth metabolism in addition to energy is provided, in lactic acid
Accessory substance lactic acid can be generated under the catalytic action of dehydrogenase.Lactic acid just has certain toxic action to cell in itself, in addition, raw
Into lactic acid can reduce the pH of culture environment, pH need to add lye in order to control, and then cause osmotic pressure to be sharply increased, and influence indirectly
The growth of cell and the expression of product.In the present invention, by controlling the amount of glucose in nutrient solution and reducing cultivation temperature, greatly
The generation and accumulation of accessory substance lactic acid are reduced greatly, and phase lactic acid maintains reduced levels always after incubation.(relate in the prior art
And (such as patent CN105189735 A add divalent transition metal salt to reduce breast in cell culture for the operation of reduction lactic acid
The accumulation of acid) but many because being known as of lactic acid generation and accumulation can be influenced, what is emphasized herein is exactly the amount and drop of glucose
The influence that low cultivation temperature generates lactic acid and accumulates, for glucose, appropriate concentration of glucose, can obtain cell growth
To improvement, cell is improved the utilization rate of glucose, if concentration of glucose is too low, cell needed nutrient matter is not
Foot, if concentration of glucose is excessive, the accumulation of accessory substance lactic acid will increase.
(4) target protein content：Cell culture processes provided by the invention, in 200L bioreactors, during rewinding
The content of destination protein is more than 4g/L, better than existing extensive CHO fermentation techniques 2-3g/L.
(5) stability between batch：The coefficient of variation (Coefficient of Variation) be initial data standard deviation and
The ratio of initial data average, can eliminate the influence of measurement scale and dimension, carry out objective comparison.The coefficient of variation is measurement data
In each observation degree of variation statistic, in general, the coefficient of variation is bigger, and the dispersion degree of each observation is bigger, more not
Stablize.When carrying out data statistic analysis, if the coefficient of variation is more than 10%, to consider that the data are abnormal.The present invention carries
The cell culture processes of confession, three batches of 200L creation datas show (see embodiment 9~11), and destination protein content is respectively 4.33g/
L, 4.20g/L, 4.15g/L, coefficient of variation CV are 2.2%；Cell viability is respectively 86.2%, 86.9%, 84.7%, variation lines
Number CV is 1.3%；Highest viable cell density is 37.25 × 106cells/ml、39.66×106cells/ml、37.08×
106Cells/ml, coefficient of variation CV are 3.8%.Therefore, cell culture data is produced as it can be seen that cell by the 200L of three batches
Growth tendency, motility rate variation tendency, the expression quantity of the concentration variation tendency of accessory substance and destination protein all maintain well
Stability between uniformity and batch, is adapted to amplification production.
Mass cell culture is carried out using commercialization culture medium in my company's technique, it is contemplated that commercial company can be with stringent
Quality control standard carry out culture medium preparation, it is ensured that the stability between batch, has stability advantage.
Brief description of the drawings：
Fig. 1：The lactate level of embodiment 1-5 and comparative example 1-2；
Fig. 2：The destination protein content of embodiment 1-5 and comparative example 1-2；
Fig. 3：The lactate level of embodiment 9-11；
Fig. 4：The destination protein content of embodiment 9-11.
Embodiment
The following examples are only used for further illustrating the present invention but are not limited to the present invention.It is all to be based on the above of the present invention
The technology realized belongs to the scope of the invention.
In embodiments of the present invention, cell culture is purchased from gibco companies with basal medium CD Forti CHO,
Poloxamer 188 is purchased from Hyclone companies, and M20A is purchased from Shanghai De Siteli Bioisystech Co., Ltd；Cell culture is used
Supplemented medium F001S, F001B are purchased from Shanghai De Siteli Bioisystech Co., Ltd.
CD FortiCHO article No.s：A14286EJ；M20A article No.s：670226；F001S article No.s：670227；F001B article No.s：
670228；188 article No.s of poloxamer：1.37065.1000.
Embodiment 1
Shaking culture
The preparation of basal medium：
0.86g CD FortiCHO, 0.175g glucose, 0.035g poloxamer 188 is taken to be configured to solution A, wherein
The concentration that the concentration of CD FortiCHO is 24.56g/L, the concentration of glucose is 5g/L, poloxamer 188 is 1g/L.
0.96g M20A are taken to be configured to the solution B that concentration is 27.62g/L.
35ml solution As are mixed into obtain basal medium with 35ml solution Bs.
The preparation of supplemented medium：
4.45g F001S are taken to be configured to the solution C (27ml) that concentration is 164.9g/L.
0.2g F001B are taken to be configured to the solution D (2.7ml) that concentration is 76g/L.
In cell cultivation process, solution C and solution D are as supplemented medium, and by volume 10:1 squeezes into shaking flask respectively.
Recovery cell, CD FortiCHO are inoculated into after amplification by cell dilution:M20A=1:In 1 basal medium, rise
Initial body accumulates 70ml, at 36-38 DEG C, 7.0% ± 1.0%CO2, cultivate under the conditions of 110rpm ± 10rpm, when viable cell density reaches
(12-18)×106During cells/ml, temperature is adjusted to 33-34 DEG C and carries out cooling culture.With F001S:F001B=10:1 is
Supplemented medium, carries out feed-batch culture, control concentration of glucose is in 4g/L~8g/L according to concentration of glucose change in nutrient solution.
The volume fraction of basal medium is 70.2%；The volume fraction of supplemented medium is 29.8%.
It the results are shown in Table 4-5, attached drawing 1, attached drawing 2.
4 viable count unit of table：×106cells/ml
| Number of days | Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 | Comparative example 1 | Comparative example 2 |
| 0 | 0.44 | 0.4 | 0.37 | 0.44 | 0.42 | 0.3 | 0.3 |
| 1 | 0.89 | 0.78 | 0.79 | 1.02 | 0.87 | 0.59 | 0.61 |
| 2 | 2.31 | 2.12 | 2.11 | 2.83 | 2.35 | 1.07 | 1.82 |
| 3 | 6.77 | 5.83 | 5.84 | 6.89 | 6.59 | 2.18 | 4.05 |
| 4 | 10.41 | 8.4 | 8.75 | 10.78 | 10.81 | 4.06 | 7.78 |
| 5 | 20.47 | 15.34 | 14.52 | 22.54 | 21.53 | 5.95 | 9.83 |
| 6 | 29.19 | 20.15 | 20.56 | 30.69 | 29.89 | 9 | 13.51 |
| 7 | 32.89 | 22.31 | 27.32 | 34.56 | 33.56 | 11.01 | 16.23 |
| 8 | 31.8 | 24.1 | 25.41 | 32.45 | 32.52 | 11.48 | 15.67 |
| 9 | 30.37 | 23.87 | 24.61 | 31.85 | 30.89 | 11.2 | 17.15 |
| 10 | 26.88 | 19.38 | 20 | 28.56 | 26.32 | 12.18 | 16.1 |
| 11 | 24.11 | 17.45 | 19.04 | 25.41 | 25.84 | 11.13 | 15.03 |
Table 5
Cell viability unit：%
Embodiment 2
Shaking culture
The preparation of basal medium：
0.69g CD FortiCHO, 0.14g glucose, 0.028g poloxamer 188 is taken to be configured to solution A, wherein
The concentration that the concentration of CD FortiCHO is 24.56g/L, the concentration of glucose is 5g/L, poloxamer 188 is 1g/L.
1.16g M20A are taken to be configured to the solution B that concentration is 27.62g/L.
28ml solution As are mixed into obtain basal medium with 42ml solution Bs.
The preparation of supplemented medium：
4.45g F001S are taken to be configured to the solution C (27ml) that concentration is 164.9g/L.
0.2g F001B are taken to be configured to the solution D (2.7ml) that concentration is 76g/L.
In cell cultivation process, solution C and solution D are as supplemented medium, and by volume 10:1 squeezes into shaking flask respectively.
Recovery cell, CD FortiCHO are inoculated into after amplification by cell dilution:M20A=1:In 1.5 basal mediums,
Initial volume 70ml, at 36-38 DEG C, 7.0% ± 1.0%CO2, cultivate under the conditions of 110rpm ± 10rpm, when viable cell density reaches
To (12-18) × 106During cells/ml, temperature is adjusted to 33-34 DEG C and carries out cooling culture.With F001S:F001B=10:1
For supplemented medium, feed-batch culture is carried out according to concentration of glucose change in nutrient solution, control concentration of glucose is in 4g/L~8g/
L.The volume fraction of basal medium is 70.2%；The volume fraction of supplemented medium is 29.8%.
It the results are shown in Table 4-5, attached drawing 1, attached drawing 2.
Embodiment 3
Shaking culture
The preparation of basal medium：
1.075g CD FortiCHO, 0.22g glucose, 0.044g poloxamer 188 is taken to be configured to solution A, wherein
The concentration that the concentration of CD FortiCHO is 24.56g/L, the concentration of glucose is 5g/L, poloxamer 188 is 1g/L.
0.725g M20A are taken to be configured to the solution B that concentration is 27.62g/L.
43.75ml solution As are mixed into obtain basal medium with 26.25ml solution Bs.
The preparation of supplemented medium：
4.12g F001S are taken to be configured to the solution C (25ml) that concentration is 164.9g/L.
0.38g F001B are taken to be configured to the solution D (5ml) that concentration is 76g/L.
In cell cultivation process, solution C and solution D are as supplemented medium, and by volume 5:1 squeezes into shaking flask respectively.
Recovery cell, CD FortiCHO are inoculated into after amplification by cell dilution:M20A=1:In 0.6 basal medium,
Initial volume 70ml, at 36-38 DEG C, 7.0% ± 1.0%CO2, cultivate under the conditions of 110rpm ± 10rpm, when viable cell density reaches
To (12-18) × 106During cells/ml, temperature is adjusted to 33-34 DEG C and carries out cooling culture.With F001S:F001B=5:1 is
Supplemented medium, carries out feed-batch culture, control concentration of glucose is in 4g/L~8g/L according to concentration of glucose change in nutrient solution.
The volume fraction of basal medium is 70%；The volume fraction of supplemented medium is 30%.
It the results are shown in Table 4-5, attached drawing 1, attached drawing 2.
Embodiment 4
Shaking culture
The preparation of basal medium：
0.86g CD FortiCHO, 0.175g glucose, 0.035g poloxamer 188 is taken to be configured to solution A, wherein
The concentration that the concentration of CD FortiCHO is 24.56g/L, the concentration of glucose is 5g/L, poloxamer 188 is 1g/L.
0.96g M20A are taken to be configured to the solution B that concentration is 27.62g/L.
35ml solution As are mixed into obtain basal medium with 35ml solution Bs.
The preparation of supplemented medium：
4.947g F001S are taken to be configured to the solution C (30ml) that concentration is 164.9g/L.
0.152g F001B are taken to be configured to the solution D (2ml) that concentration is 76g/L.
In cell cultivation process, solution C and solution D are as supplemented medium, and by volume 15:1 squeezes into shaking flask respectively.
Recovery cell, CD FortiCHO are inoculated into after amplification by cell dilution:M20A=1:In 1 basal medium, rise
Initial body accumulates 70ml, at 36-38 DEG C, 7.0% ± 1.0%CO2, cultivate under the conditions of 110rpm ± 10rpm, when viable cell density reaches
(12-18)×106During cells/ml, temperature is adjusted to 33-34 DEG C and carries out cooling culture.With F001S:F001B=15:1 is
Supplemented medium, carries out feed-batch culture, control concentration of glucose is in 4g/L~8g/L according to concentration of glucose change in nutrient solution.
The volume fraction of basal medium is 68.6%；The volume fraction of supplemented medium is 31.4%.
It the results are shown in Table 4-5, attached drawing 1, attached drawing 2.
Embodiment 5
Shaking culture
The preparation of basal medium：
0.98g CD FortiCHO, 0.2g glucose, 0.04g poloxamer 188 is taken to be configured to solution A, wherein CD
The concentration that the concentration of FortiCHO is 24.56g/L, the concentration of glucose is 5g/L, poloxamer 188 is 1g/L.
0.88g M20A are taken to be configured to the solution B that concentration is 27.62g/L.
40ml solution As are mixed into obtain basal medium with 32ml solution Bs.
The preparation of supplemented medium：
4.53g F001S are taken to be configured to the solution C (27.5ml) that concentration is 164.9g/L.
0.19g F001B are taken to be configured to the solution D (2.5ml) that concentration is 76g/L.
In cell cultivation process, solution C and solution D are as supplemented medium, and by volume 11:1 squeezes into shaking flask respectively.
Recovery cell, CD FortiCHO are inoculated into after amplification by cell dilution:M20A=1:In 0.8 basal medium,
Initial volume 72ml, at 36-38 DEG C, 7.0% ± 1.0%CO2, cultivate under the conditions of 110rpm ± 10rpm, when viable cell density reaches
To (12-18) × 106During cells/ml, temperature is adjusted to 33-34 DEG C and carries out cooling culture.With F001S:F001B=11:1
For supplemented medium, feed-batch culture is carried out according to concentration of glucose change in nutrient solution, control concentration of glucose is in 4g/L~8g/
L.The volume fraction of basal medium is 70.6%；The volume fraction of supplemented medium is 29.4%.
It the results are shown in Table 4-5, attached drawing 1, attached drawing 2.
1~embodiment of embodiment 5 is compared by changing the volume ratio of basal medium and the volume ratio of supplemented medium
Compared with from highest viable cell density, embodiment 1, embodiment 4 and 5 result of embodiment are best, other to take second place；Cell viability
Aspect, the Cell viability in cell cultivation process under five kinds of condition of culture is always maintained at well, when terminating to cultivate the 12nd
Its Cell viability is all higher than 80%；Analyzed from the change of lactic acid, five kinds of condition of culture variation tendencies are similar, Initial stage of culture product
Tired, late stage of culture is gradually reduced, and the lactic acid content of embodiment 4 is higher at the end of culture, and the lactic acid content of remaining embodiment is equal
It can control in relatively low scope.From destination protein containing taking temperature, 1 highest of embodiment, embodiment 2 and embodiment 5 are taken second place.Consider
Various factors, determines CD FortiCHO:M20A=1:1, F001S:F001B=10:1 is more suitable for the joint of technique amplification
Culture medium.
Note：Following embodiments, using optimal volume ratio, i.e., with CD FortiCHO:M20A=1:1 is basic culture medium,
With F001S:F001B=10:1 is supplemented medium.
Embodiment 6
2L reactors
The preparation of basal medium：
9.82g CD FortiCHO, 2.4g glucose, 0.48g poloxamer 188 is taken to be configured to solution A, wherein CD
The concentration that the concentration of FortiCHO is 24.56g/L, the concentration of glucose is 6g/L, poloxamer 188 is 1.2g/L.
11.05g M20A are taken to be configured to the solution B that concentration is 27.62g/L
400ml solution As are mixed into obtain basal medium with 400ml solution Bs.
The preparation of supplemented medium：
44.52g F001S are taken to be configured to the solution C (270ml) that concentration is 164.9g/L
2.05g F001B are taken to be configured to the solution D (27ml) that concentration is 76g/L
In cell cultivation process, solution C and solution D are as supplemented medium, and by volume 10:1 squeezes into reactor respectively
It is interior.
With CD FortiCHO:M20A=1:1 basal medium, each control parameter of bioreactor are arranged to：Reactor pH
Control range 7.0 ± 0.2, dissolved oxygen：40.0%th, initial speed of agitator 180rpm ± 10rpm, deep ventilation flow velocity：25.0ccm,
36-38 DEG C of initial incubation temperature, when viable cell density reaches (12~18) × 106During cell/ml, 33-34 DEG C is cooled to.Using
F001S:F001B=10:1 feed supplement stream adds system to carry out feed supplement fed-batch cultivation, and cultivation cycle about 12 to 14 days, lives according to cell
Rate is not less than 80% principle, harvests culture supernatant.Concentration of glucose is in 4g/L~8g/L, decision feed supplement in control nutrient solution
Stream dosage.The volume fraction of basal medium is 72.9%；The volume fraction of supplemented medium is 27.1%.
As a result：Highest viable cell density reaches 27.95 × 106cells/ml；The Cell viability one in whole incubation
Directly maintain in very high level, more than 90% is stilled remain in Cell viability during the 12nd day harvest；From the change of lactic acid
Analysis, slowly accumulated in the early period of culture, after incubation the phase be consumed, to harvest the 12nd day when, the concentration of lactic acid drops to
0.5g/L or so.The change of metabolic by-product shows that technique will not be produced in follow-up amplification because of the accumulation of metabolic by-product
Raw risk；From the point of view of destination protein content, reactor destination protein in final harvest reaches 4.17g/L.
Embodiment 7
10L reactors
The preparation of basal medium：
61.4g CD FortiCHO, 10g glucose, 2.37g poloxamer 188 is taken to be configured to solution A, wherein CD
The concentration that the concentration of FortiCHO is 24.56g/L, the concentration of glucose is 4g/L, poloxamer 188 is 0.95g/L.
69.05g M20A are taken to be configured to the solution B that concentration is 27.62g/L
2.5L solution As are mixed into obtain basal medium with 2.5L solution Bs.
The preparation of supplemented medium：
362.78g F001S are taken to be configured to the solution C (2.2L) that concentration is 164.9g/L
16.72g F001B are taken to be configured to the solution D (0.22L) that concentration is 76g/L
In cell cultivation process, solution C and solution D are as supplemented medium, and by volume 10:1 squeezes into reactor respectively
It is interior.
With CD FortiCHO:M20A=1:1 basal medium, with F001S:F001B=10:1 feed supplement stream add system into
Row feed supplement fed-batch cultivation, Initial seeding density are (3-7) × 105Cell/ml, when viable cell density reach (12~18) ×
106During cell/ml, cultivation temperature is adjusted to 33-34 DEG C by 36-38 DEG C, top layer throughput is 0.05lpm, deep ventilation amount
For 0.05lpm, cultivation cycle about 12-14 days.The volume fraction of basal medium is 67.4%；The volume fraction of supplemented medium
For 32.6%.
As a result：Culture 13 days, highest viable cell density 36.54 × 106cells/ml；Cell viability remains at during harvest
More than 85%, it is 88.5%；Lactic acid, accumulates in Initial stage of culture, and the later stage starts to be gradually reduced, and the concentration of lactic acid drops to during harvest
1.02g/L；Target protein content 5.33g/L.
Embodiment 8
10L reactors
The preparation of basal medium：
73.68g CD FortiCHO, g glucose, 2.76g poloxamer 188 is taken to be configured to solution A, wherein CD
The concentration that the concentration of FortiCHO is 24.56g/L, the concentration of glucose is 7g/L, poloxamer 188 is 0.92g/L.
82.86g M20A are taken to be configured to the solution B that concentration is 27.62g/L
3L solution As are mixed into obtain basal medium with 3L solution Bs.
The preparation of supplemented medium：
329.8g F001S are taken to be configured to the solution C (2L) that concentration is 164.9g/L.
15.2g F001B are taken to be configured to the solution D (0.2L) that concentration is 76g/L
In cell cultivation process, solution C and solution D are as supplemented medium, and by volume 10:1 squeezes into reactor respectively
It is interior.
With CD FortiCHO:M20A=1:1 basal medium, with F001S:F001B=10:1 feed supplement stream add system into
Row feed supplement fed-batch cultivation, Initial seeding density are (3-7) × 105Cell/ml, when viable cell density reach (12~18) ×
106During cell/ml, cultivation temperature is adjusted to 33-34 DEG C by 36-38 DEG C, top layer throughput is 0.05lpm, deep ventilation amount
For 0.05lpm, cultivation cycle about 12-14 days.The volume fraction of basal medium is 73.2%；The volume fraction of supplemented medium
For 26.8%.
As a result：Culture 13 days, highest viable cell density 30.89 × 106cells/ml；Cell viability remains at during harvest
More than 85%, it is 87.6%；Lactic acid, accumulates in Initial stage of culture, and the later stage starts to be gradually reduced, and the concentration of lactic acid drops to during harvest
1.23g/L；Target protein content 5.00g/L.
In two 10L reactor process amplification tests, growth tendency, motility rate variation tendency, the accessory substance lactic acid of cell
Concentration variation tendency and the expression quantity of destination protein all maintain good uniformity, and flask process and 2L reactor works
Skill is consistent.Illustrate 10L bioreactor culture techniques it is with good stability and can amplification, be adapted for more massive
Amplification production.
Embodiment 9
200L reactors
The preparation of basal medium：
687.68g CD FortiCHO, 112g glucose, 33.6g poloxamer 188 is taken to be configured to solution A, wherein
The concentration that the concentration of CD FortiCHO is 24.56g/L, the concentration of glucose is 4g/L, poloxamer 188 is 1.2g/L.
773.36g M20A are taken to be configured to the solution B that concentration is 27.62g/L
28L solution As are mixed into obtain basal medium with 28L solution Bs.
The preparation of supplemented medium：
3627.8g F001S are taken to be configured to the solution C (22L) that concentration is 164.9g/L.
167.2g F001B are taken to be configured to the solution D (2.2L) that concentration is 76g/L.
In cell cultivation process, solution C and solution D are as supplemented medium, and by volume 10:1 squeezes into reactor respectively
It is interior.
With CD FortiCHO:M20A=1:1 is basic culture medium, with F001S:F001B=10:1 is supplemented medium,
Initial seeding density 7 × 105Cell/ml, after cultivating 24h, according to the concentration of glucose in culture medium, adds supplemented medium,
Concentration of glucose is 36.7 DEG C in 6g/L, progress Fed-Batch cultures, cultivation temperature in control nutrient solution.Work as viable cell density
Reach 18 × 106Cell/ml, 33.5 DEG C are down to by cultivation temperature.Other culture parameters are：PH 7.0 ± 0.2, dissolved oxygen
40.0%th, initial speed 90rpm, top layer throughput are 0.5lpm, and deep ventilation amount is 0.5lpm, and incubation time is 12 × 24h.
The volume fraction of basal medium is 69.8%；The volume fraction of supplemented medium is 30.2%.
The result is shown in attached drawing 3, attached drawing 4, table 6-7.
Table 6
Viable count unit：×106cells/ml
| Number of days | Embodiment 9 | Embodiment 10 | Embodiment 11 |
| 0 | 0.67 | 0.59 | 0.61 |
| 1 | 1.7 | 1.82 | 1.63 |
| 2 | 4.08 | 4.76 | 4.29 |
| 3 | 10.16 | 13.59 | 11.11 |
| 4 | 17.36 | 17.01 | 17.45 |
| 5 | 22.15 | 24.03 | 24.74 |
| 6 | 31.22 | 32.27 | 32.02 |
| 7 | 35.3 | 37.79 | 36.04 |
| 8 | 36.28 | 38.23 | 37.08 |
| 9 | 37.25 | 39.66 | 34.44 |
| 10 | 34.97 | 34.32 | 32.41 |
| 11 | 33.18 | 32.03 | 31.19 |
| 12 | 29.57 | 31 | 27.96 |
Table 7
Cell viability unit：%
Embodiment 10
200L reactors
The preparation of basal medium：
736.8g CD FortiCHO, 198g glucose, 33g poloxamer 188 is taken to be configured to solution A, wherein CD
The concentration that the concentration of FortiCHO is 24.56g/L, the concentration of glucose is 6.6g/L, poloxamer 188 is 1.1g/L.
828.6g M20A are taken to be configured to the solution B that concentration is 27.62g/L
30L solution As are mixed into obtain basal medium with 30L solution Bs.
The preparation of supplemented medium：
4452.3g F001S are taken to be configured to the solution C (27L) that concentration is 164.9g/L.
205.2g F001B are taken to be configured to the solution D (2.7L) that concentration is 76g/L.
In cell cultivation process, solution C and solution D are as supplemented medium, and by volume 10:1 squeezes into reactor respectively
It is interior.
With CD FortiCHO:M20A=1:1 is basic culture medium, with F001S:F001B=10:1 is supplemented medium,
Initial seeding density 7 × 105Cell/ml, after cultivating 24h, according to the concentration of glucose in culture medium, adds supplemented medium,
Concentration of glucose is 36.7 DEG C in 6g/L, progress Fed-Batch cultures, cultivation temperature in control nutrient solution.Work as viable cell density
Reach 18 × 106Cell/ml, 33.5 DEG C are down to by cultivation temperature.Other culture parameters are：PH 7.0 ± 0.2, dissolved oxygen
40.0%th, initial speed 90rpm, top layer throughput are 0.5lpm, and deep ventilation amount is 0.5lpm, and incubation time is 12 × 24h.
The volume fraction of basal medium is 66.9%；The volume fraction of supplemented medium is 33.1%.
The result is shown in attached drawing 3, attached drawing 4, table 6-7.
Embodiment 11
200L reactors
The preparation of basal medium：
761.36g CD FortiCHO, 241.8g glucose, 30.38g poloxamer 188 is taken to be configured to solution A, its
The concentration that the concentration of middle CD FortiCHO is 24.56g/L, the concentration of glucose is 7.8g/L, poloxamer 188 is
0.98g/L。
856.22g M20A are taken to be configured to the solution B that concentration is 27.62g/L
31L solution As are mixed into obtain basal medium with 31L solution Bs.
The preparation of supplemented medium：
3133.1g F001S are taken to be configured to the solution C (19L) that concentration is 164.9g/L.
144.4g F001B are taken to be configured to the solution D (1.9L) that concentration is 76g/L.
In cell cultivation process, solution C and solution D are as supplemented medium, and by volume 10:1 squeezes into reactor respectively
It is interior.
With CD FortiCHO:M20A=1:1 is basic culture medium, with F001S:F001B=10:1 is supplemented medium,
Initial seeding density 7 × 105Cell/ml, after cultivating 24h, according to the concentration of glucose in culture medium, adds supplemented medium,
Concentration of glucose is 36.7 DEG C in 6g/L, progress Fed-Batch cultures, cultivation temperature in control nutrient solution.Work as viable cell density
Reach 18 × 106Cell/ml, 33.5 DEG C are down to by cultivation temperature.Other culture parameters are：PH 7.0 ± 0.2, dissolved oxygen
40.0%th, initial speed 90rpm, top layer throughput are 0.5lpm, and deep ventilation amount is 0.5lpm, and incubation time is 12 × 24h.
The volume fraction of basal medium is 74.8%；The volume fraction of supplemented medium is 25.2%.
The result is shown in attached drawing 3, attached drawing 4, table 6-7.
As a result：In the 200L reactors experiment of three batches, growth tendency, motility rate variation tendency, the accessory substance of cell
Concentration variation tendency and the expression quantity of destination protein all maintain good uniformity, and protected with small-scale technique before
Hold consistent.Illustrate that 200L bioreactor culture techniques are with good stability, be adapted to amplification production.
Comparative example 1
Shaking culture
The preparation method of basal medium：
Take 1.72g CD FortiCHO to be configured to the solution A that concentration is 24.56g/L, be basal medium (70ml).
The preparation method of supplemented medium：
4.45g F001S are taken to be configured to the solution B (27ml) that concentration is 164.9g/L.
0.2g F001B are taken to be configured to the solution C (2.7ml) that concentration is 76g/L.
In cell cultivation process, solution B and solution C are as supplemented medium, and by volume 10:1 squeezes into shaking flask respectively.
Cell dilution, is inoculated into CD FortiCHO basal mediums, initial volume by recovery cell after amplification
70ml, at 36-38 DEG C, 7.0% ± 1.0%CO2, cultivate under the conditions of 110rpm ± 10rpm, when viable cell density reaches (12-
18)×106During cells/ml, temperature is adjusted to 33-34 DEG C and carries out cooling culture.With F001S:F001B=10:1 is feed supplement
Culture medium, carries out feed-batch culture, control concentration of glucose is in 4g/L~8g/L according to concentration of glucose change in nutrient solution.Basis
The volume fraction of culture medium is 70.2%；The volume fraction of supplemented medium is 29.8%.
It the results are shown in Table 4-5 1-2, attached drawing 1, attached drawing 2.
Comparative example 2
Shaking culture
The preparation method of basal medium：
Take 1.93g M20A to be configured to the solution A that concentration is 27.62g/L, be basal medium (70ml).
The preparation method of supplemented medium：
4.45g F001S are taken to be configured to the solution B (27ml) that concentration is 164.9g/L.
0.2g F001B are taken to be configured to the solution C (2.7ml) that concentration is 76g/L.
In cell cultivation process, solution B and solution C are as supplemented medium, and by volume 10:1 squeezes into shaking flask respectively.
Cell dilution, is inoculated into CD FortiCHO basal mediums, initial volume by recovery cell after amplification
70ml, at 36-38 DEG C, 7.0% ± 1.0%CO2, cultivate under the conditions of 110rpm ± 10rpm, when viable cell density reaches (12-
18)×106During cells/ml, temperature is adjusted to 33-34 DEG C and carries out cooling culture.With F001S:F001B=10:1 is feed supplement
Culture medium, carries out feed-batch culture, control concentration of glucose is in 4g/L~8g/L according to concentration of glucose change in nutrient solution.Basis
The volume fraction of culture medium is 70.2%；The volume fraction of supplemented medium is 29.8%.
It the results are shown in Table 4-5, attached drawing 1, attached drawing 2.
As a result：Embodiment 1, comparative example 1, comparative example 2 are compared by changing basal medium species, are lived from highest thin
From the point of view of in born of the same parents' density, embodiment 1 will be significantly higher than comparative example 1 and comparative example 2；In terms of Cell viability, under three kinds of condition of culture
Cell viability be always maintained at well, when terminating to cultivate, the 12nd day Viability is all higher than 80%, and wherein comparative example 1 is slightly
Height, secondly embodiment 1, comparative example 2 is minimum；To be analyzed from the change of lactic acid, embodiment 1 is similar to 2 variation tendency of comparative example,
Initial stage of culture accumulates, and late stage of culture is gradually reduced, but the lactic acid content of comparative example 2 is higher at the end of culture, implements 1 lactic acid and contains
Amount can be controlled in relatively low scope, though contrast 1 lactic acid content it is relatively low at the end of culture, its variation tendency is simultaneously unstable；
From destination protein containing taking temperature, embodiment 1 will be significantly higher than comparative example 1 and comparative example 2.Therefore, commbined foundations culture medium is better than
One of which basal medium is used alone.
Embodiment described above only expresses the several embodiments of the present invention, its description is more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention cannot be interpreted as, it is noted that for those skilled in the art,
Without departing from the inventive concept of the premise, some deformations and transformation can also be made, these belong to the protection model of the present invention
Enclose, therefore, the protection domain of patent of the present invention is determined by the appended claims.
Claims (10)
- A kind of 1. Combined culture base for expressing adalimumab, it is characterised in that it is made of basal medium and supplemented medium, The percentage by volume of basal medium and supplemented medium is respectively：55%-75% and 25%-45%；Wherein basal medium is made of solution A and solution B, solution A:The volume ratio of B is 1：In 0.5-1.5, wherein solution A into It is divided into 20-28g/L CD FortiCHO, 0.9-1.2g/L poloxamer 188 and 4-8g/L glucose, remaining component is water； Component is 20-28g/L M20A in solution B, remaining component is water；Wherein supplemented medium is made of solution C and solution D, solution C:The volume ratio of D is 5-15：1, wherein component in solution C For 150-200g/L F001S, remaining component is water；Component is 50-100g/L F001B wherein in solution D, remaining component is Water.
- 2. Combined culture base according to claim 1, it is characterised in that the body of the basal medium and supplemented medium Accumulating percentage is respectively：65%-75% and 25%-35%.
- 3. Combined culture base according to claim 1, it is characterised in that the solution A：The volume ratio of B is 1:1.
- 4. Combined culture base according to claim 1, it is characterised in that component is 24.56g/L CD in the solution A FortiCHO, 1g/L poloxamer 188 and 5g/L glucose, remaining component are water；Solution B is 27.62g/L M20A, its Remaining component is water.
- 5. Combined culture base according to claim 1, it is characterised in that the solution C：The volume ratio of D is 10：1.
- 6. Combined culture base according to claim 1, it is characterised in that component is 164.9g/L's in the solution C F001S, remaining component are water；Component is 76g/L F001B wherein in solution D, remaining component is water.
- 7. the preparation method of the Combined culture base described in a kind of claim 1, it is characterised in that comprise the steps of：1) CD FortiCHO solid mediums are weighed, poloxamer188 and glucose are dissolved in the water, and are configured to solution A；2) M20A solid mediums are weighed, are dissolved in the water, are configured to solution B；3) by solution A and B with 1:The volume ratio mixing of 0.5-1.5, obtains basal medium；4) F001S and F001B are weighed, is dissolved in water, is configured to solution C and D, by solution C and D according to volume ratio 5- 15:1 composition supplemented medium, solution C and D cannot be pre-mixed at this time；5) basal medium has collectively constituted Combined culture base with supplemented medium described in step 4) in step 3).
- 8. a kind of adalimumab cell culture processes using Combined culture base described in claim 1, it is characterised in that described Cell culture processes comprise the steps of：A) adalimumab cell line is inoculated with into the basal medium, Initial seeding density is 3-7 × 105cells/ml； After when culture 12-36 is small at 36-38 DEG C of temperature, according to the concentration 4-8g/L of glucose in basal medium, feed-batch culture is added Base, feed postition add for stream, and stream dosage ensures concentration of glucose and the stabilization of other nutrient levels；B) when viable cell density reaches 12-18 × 106Cells/ml, 33-34 DEG C is down to by cultivation temperature；C) other culture parameters are：PH 7.0 ± 0.2, dissolved oxygen 30-50.0%, initial speed 60-100rpm, top layer throughput are 0.2-1lpm, deep ventilation amount are 0.2-1lpm, after cultivating 10-15*24h, obtain the adalimumab cell culture fluid, into Row monoclonal antibody purification step, obtains the adalimumab.
- 9. application according to claim 8, it is characterised in that the expression cell is mammalian cell, and preferably CHO is thin Born of the same parents.
- 10. application according to claim 8, it is characterised in that the flow acceleration is 600~800ml/min.
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| CN110343666A (en) * | 2019-07-10 | 2019-10-18 | 通化东宝生物科技有限公司 | A kind of supplemented medium and its preparation method and application of Chinese hamster ovary celI culture |
| CN113567678A (en) * | 2020-04-28 | 2021-10-29 | 信达生物制药(苏州)有限公司 | Method for evaluating stability of culture medium dry powder |
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| CN104004814A (en) * | 2013-02-21 | 2014-08-27 | 江苏先声药物研究有限公司 | Method for precisely controlling monoclonal antibody product quality through animal cell fed-batch cultivation |
| CN104212769A (en) * | 2014-07-14 | 2014-12-17 | 北京益生合生物科技有限公司 | Cell culture medium additive used for highly producing monoclonal antibody |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101663390A (en) * | 2006-09-13 | 2010-03-03 | 艾博特公司 | Cell culture improvements |
| CN104004814A (en) * | 2013-02-21 | 2014-08-27 | 江苏先声药物研究有限公司 | Method for precisely controlling monoclonal antibody product quality through animal cell fed-batch cultivation |
| CN104212769A (en) * | 2014-07-14 | 2014-12-17 | 北京益生合生物科技有限公司 | Cell culture medium additive used for highly producing monoclonal antibody |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110343666A (en) * | 2019-07-10 | 2019-10-18 | 通化东宝生物科技有限公司 | A kind of supplemented medium and its preparation method and application of Chinese hamster ovary celI culture |
| CN110343666B (en) * | 2019-07-10 | 2023-05-30 | 通化东宝药业股份有限公司 | Feed supplement culture medium for CHO cell culture and preparation method and application thereof |
| CN113567678A (en) * | 2020-04-28 | 2021-10-29 | 信达生物制药(苏州)有限公司 | Method for evaluating stability of culture medium dry powder |
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