CN109385401A - A kind of cultural method of Chinese hamster ovary celI that expressing recombinant protein - Google Patents
A kind of cultural method of Chinese hamster ovary celI that expressing recombinant protein Download PDFInfo
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- CN109385401A CN109385401A CN201710682003.1A CN201710682003A CN109385401A CN 109385401 A CN109385401 A CN 109385401A CN 201710682003 A CN201710682003 A CN 201710682003A CN 109385401 A CN109385401 A CN 109385401A
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- 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
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/005—Glycopeptides, glycoproteins
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
Abstract
The invention belongs to protein medical bioengineering and technical fields, disclose a kind of cultural method of Chinese hamster ovary celI for expressing recombinant protein, sialic acid is added in cell cultivation process.Experiment shows that the present invention adds sialic acid in cell cultivation process and the sialylation levels of expression recombinant protein can be improved.And the substrate of the inherently sialylated process of sialic acid, the sialic acid of addition can be added to the least significant end of protein glycosylation by enzymatic reaction, will not bring risk, and price is relatively low, may be implemented to use in large-scale production.
Description
Technical field
The invention belongs to protein medical bioengineering and technical fields, and in particular to a kind of CHO for expressing recombinant protein
The cultural method of cell, especially a kind of cultural method of the Chinese hamster ovary celI for the sialylation levels for improving expression recombinant protein.
Background technique
Biological products have become the important component in treatment medication market at present.Treatment biological products are main at present
Including antibody class, cytokine class and polypeptide (such as various insulin preparations), these biological products are all to pass through genetic recombination
Technology is expressed by different hosts cell or host strain.Wherein antibody class and most cytokine class products are moved by lactation
Obtained after the expression of object cell through purifying because these drugs all have it is glycosylation modified.
Glycosylation modified biological activity and stability to antibody or cell factor kind biological product plays a significant role,
For example the ADCC effect of antibody is mainly exactly to be mediated by its Fc sections of sugar chain.Glycosylation modified is an extremely complex mistake
Journey, the type of glycosylation of different protein moleculars is all different in addition same protein molecular on sugar chain on different glycosylation site
Also different, and there are inhomogeneities.Since sugar chain is to the importance and sugar chain sheet of protein molecular medicines structure and function
The high complexity of body, glycosylation control become very important a part in protein drug production and quality control.
Type of glycosylation includes many kinds, and every kind of sugar chain type can all generate different influences to protein drug, such as high
Mannose-modified can reduce protein drug in people's intracorporal half-life period, and the sialylation levels of sugar chain molecule least significant end can extend
The half-life period of protein drug, these all have a huge impact the pharmacokinetics PK of drug.Most antibody drugs are only
There is a glycosylation site, and the Degree of sialylation of the sugar-chain end is all very low.Many cytokine classes and fusion protein
Class drug has multiple glycosylation sites, and the sugar chain in different loci has different degrees of sialylated, sialylation levels
Half-life period and PK to these albumen can have a huge impact, such as EPO, FSH etc..Therefore these drugs must be controlled
Sialylation levels.
It is all by cultivating mammalian cell expression, Chinese hamster ovary celI with glycosylated cell factor and fusion protein
It is wherein to use most common expression cell.In the incubation of mammalian cell in the bioreactor, culture will receive
And component, temperature, dissolved oxygen, the influence of the multiple factors such as pH, it can be made to generate different glycosylation modified type and ratio.At present
Widely used Chinese hamster ovary celI can only generate less than 5% sialylated modification under regular culture conditions, cause expression albumen
Half-life period is shorter, is unable to satisfy the requirement of clinical treatment.
Currently, the method reported in the literature for improving expressing cho cell albumen sialylation levels is mainly included in culture medium
Middle addition ManNAC, Mn ion, dexamethasone etc..But these additives are expensive, and are not easy from final drug
It removes, there are risks.
Summary of the invention
In view of this, providing a kind of CHO for expressing recombinant protein it is an object of the invention in view of the drawbacks of the prior art
The cultural method of cell, to improve the sialylation levels of expression albumen.
To achieve the purpose of the present invention, the present invention adopts the following technical scheme:
Applicant passes through the Chinese hamster ovary celI in expression Human Follicle Stimulating Hormone (FSH) and recombinant human VEGF R-Fc fusion egg
Certain density sialic acid is added in incubation, such as N-acetyl-neuraminate (NANA), can obtain sialylation levels compared with
High expression recombinant protein, sialylation levels reach actual standard.Wherein express the Chinese hamster ovary celI of Human Follicle Stimulating Hormone
Sialic acid content reaches 8-12mol/mol albumen, expresses the sialic acid content of the Chinese hamster ovary celI of recombinant human VEGF R-Fc fusion protein
Reach 8-12mol/mol albumen.Therefore the present invention provides sialic acids to improve the sialylated of expressing cho cell recombinant protein
Application in level.
Wherein, the sialic acid is N-acetyl-neuraminate, NeuGc ALPHA2-3Gal, deaminize neuraminic acid or nerve
At least one of propylhomoserin.
Further the present invention provides a kind of cultural methods of Chinese hamster ovary celI for expressing recombinant protein, in cell cultivation process
Middle addition sialic acid.
Wherein, preferably, the concentration of the sialic acid is 5~10mM.More preferably 5mM.
Sialic acid described in the cultural method of the Chinese hamster ovary celI of expression recombinant protein of the present invention is N- acetyl nerve ammonia
Acid, NeuGc ALPHA2-3Gal, deaminize at least one of neuraminic acid or neuraminic acid.It is in some embodiments N-
N acetylneuraminic acid n, in other embodiments, the sialic acid are NeuGc ALPHA2-3Gal (NANG).
The cultural method of the Chinese hamster ovary celI of expression recombinant protein of the present invention includes but is not limited to express recombination human follicle thorn
The culture of the Chinese hamster ovary celI of recombinant protein described in the Chinese hamster ovary celI or expression recombinant human VEGF R-Fc fusion protein of hormone.
In the cultural method of the Chinese hamster ovary celI of expression recombinant protein of the present invention, the sialic acid is in expression recombinant protein
Chinese hamster ovary celI secondary culture after be inoculated in expression culture medium carry out expression culture when addition.
In some embodiments, the secondary culture is specially the Chinese hamster ovary celI recovered and express recombinant protein, is seeded to expansion
In kind culture medium, 37 DEG C, 5%CO2, 140rpm shake culture, it is primary every 2 days secondary cultures, inoculum density be 5 × 105Carefully
Born of the same parents/milliliter are carried out continuously 5 times or more secondary cultures, and when cell activity is restored to 95% or more, the cell for the culture that will suspend is connect
Kind carries out expression culture into expression culture medium.
In some embodiments, the expression culture is specially that the seed cell of secondary culture is seeded to expression culture
In base, N-acetyl-neuraminate or NeuGc ALPHA2-3Gal, 33 DEG C, 5%CO are added2, 140rpm shake culture, it is continuous to cultivate
11-15 days；Period controls pH value >=6.8 of culture solution, concentration of glucose >=20mM.
In some embodiments, the Chinese hamster ovary celI of the expression culture Human Follicle Stimulating Hormone is specially by secondary culture
The Chinese hamster ovary celI seed cell of Human Follicle Stimulating Hormone be seeded in expression culture medium, add N-acetyl-neuraminate or N-
Hydroxyacetylneuraminic acid, 33 DEG C, 5%CO2, 140rpm shake culture, continuous culture 11 days；The pH value of period control culture solution >=
6.8, concentration of glucose >=20mM.
In some embodiments, the Chinese hamster ovary celI of the expression culture Human Follicle Stimulating Hormone is specially by secondary culture
Human Follicle Stimulating Hormone Chinese hamster ovary celI seed cell be seeded to expression culture medium in, add N-acetyl-neuraminate, 33
DEG C, 5%CO2, 140rpm shake culture, continuous culture 15 days；Period control pH value >=6.8 of culture solution, concentration of glucose >=
20mM。
In some embodiments, in the expression incubation, when pH value is lower than 6.8,1% is added into culture shaking flask
(v/v) 7.5%NaHCO3Solution adjusts pH；When concentration of glucose is lower than 20mM, 1% (v/v) is added into culture shaking flask
45% glucose solution.
Further, it is of the present invention expression recombinant protein Chinese hamster ovary celI cultural method, expression culture after further include
After medium centrifugal the step of supernatant liquid filtering, purifying.
In some embodiments, the centrifugation is that 800 × g is centrifuged 5min.
In some embodiments, described to be filtered into through 0.22 μm of membrane filtration.
In some embodiments, the purifying is affinitive layer purification.
As shown from the above technical solution, the present invention provides it is a kind of express recombinant protein Chinese hamster ovary celI cultural method,
Sialic acid is added in cell cultivation process.Experiment shows that the present invention adds sialic acid in cell cultivation process and table can be improved
Up to the sialylation levels of recombinant protein.And the substrate of the inherently sialylated process of sialic acid, addition sialic acid can lead to
The least significant end that enzymatic reaction is added to protein glycosylation is crossed, risk will not be brought, and price is relatively low, may be implemented extensive
It is used in production.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described.
Fig. 1 shows cell growth/table of the Chinese hamster ovary celI of 11 shaking flask culture of embodiment expression recombinant human VEGF R-Fc fusion protein
Up to curve, wherein titer is expressing quantity, and VCD is viable cell density.
Fig. 2 shows that the cell of the Chinese hamster ovary celI of 12 5L bioreactor culture of embodiment expression Human Follicle Stimulating Hormone is raw
Length/expression curve, wherein titer is expressing quantity, and VCD is viable cell density.
Fig. 3 shows the thin of the Chinese hamster ovary celI of 13 5L bioreactor culture of embodiment expression recombinant human VEGF R-Fc fusion protein
Intracellular growth/expression curve, wherein titer is expressing quantity, and VCD is viable cell density.
Fig. 4 shows that the cell of the Chinese hamster ovary celI of 14 50L bioreactor culture of embodiment expression Human Follicle Stimulating Hormone is raw
Length/expression curve, wherein titer is expressing quantity, and VCD is viable cell density.
Fig. 5 shows the Chinese hamster ovary celI of 15 50L bioreactor culture of embodiment expression recombinant human VEGF R-Fc fusion protein
Cell grows/expresses curve, and wherein titer is expressing quantity, and VCD is viable cell density.
Specific embodiment
The invention discloses a kind of cultural methods of Chinese hamster ovary celI for expressing recombinant protein.Those skilled in the art can borrow
Reflect present disclosure, is suitably modified realization of process parameters.In particular, it should be pointed out that all similar substitutions and modifications are to this field
It is it will be apparent that they are considered as being included in the present invention for technical staff.Method of the invention passes through preferable reality
Example is applied to be described, related personnel obviously can not depart from the content of present invention, in spirit and scope to method described herein
It is modified or appropriate changes and combinations, carrys out implementation and application the technology of the present invention.
For a further understanding of the present invention, below in conjunction with the embodiment of the present invention, to the technical side in the embodiment of the present invention
Case is clearly and completely described, it is clear that and described embodiments are only a part of the embodiments of the present invention, rather than all
Embodiment.Based on the embodiments of the present invention, those of ordinary skill in the art institute without making creative work
The every other embodiment obtained, shall fall within the protection scope of the present invention.
Unless otherwise specified, reagent involved in the embodiment of the present invention is commercial product, can pass through business canal
Road purchase obtains.
The Chinese hamster ovary celI of embodiment 1, shaking flask culture expression Human Follicle Stimulating Hormone
(1) culture medium and feed supplement
1. expanding species culture medium prepares: commercially available liquid CDM4CHO culture medium
2.3.5w/v%Cell 6 solution of Boost prepares: weighing 6 powder of Cell Boost of 35g, ultrapure water dissolves simultaneously
It is settled to 1L, then through 0.22 μm of filtration sterilization；
3.200mM L-Glutamine solution prepares: weighing the L-Glutamine powder of 2.92g, ultrapure water dissolution and constant volume
To 100mL, then through 0.22 μm of filtration sterilization；(L-Glutamine mole is 146.14)
4.500mM NANA solution prepares: weighing the NANA powder of 3.09g, ultrapure water dissolves and be settled to 20mL, then
Through 0.22 μm of filtration sterilization；(NANA mole is 309.27)
5. expression culture medium prepares: measuring commercially available liquid Power CHO-2 culture medium 784mL, 16mL is added and has prepared
200mM L-Glutamine solution, add 6 solution of 3.5w/v%Cell Boost that 200mL has been prepared, after mixing
For use；(whole process carries out in Biohazard Safety Equipment, guarantees sterile)
6.45% glucose solution prepares: weighing the glucose powder of 45g, ultrapure water dissolves and be settled to 100mL, then
Through 0.22 μm of filtration sterilization；
7.7.5%NaHCO3Solution prepares: weighing the NaHCO of 7.5g3Powder, ultrapure water dissolve and are settled to 100mL, so
By 0.22 μm of filtration sterilization.
(2) instrument and equipment:
1.Thermo/371CO2Cell incubator
2.Infors/Celltron magnetic drives concussion shaking table
3. Su Jing is safe and sound/BSC-1300IIA2 Biohazard Safety Equipment
4.Roche/Cedex Bio biochemical process analyzer
The general water bath of 5.Vivo/VWB-9
The micro pH meter of 6.METTLER/S-210
7.BECKMAN/Vi-Cell XR cell counter
(3) incubation:
Subculture stage: the Chinese hamster ovary celI of one expression Human Follicle Stimulating Hormone of recovery is seeded to equipped with 20mL expanding species
In the 125mL shaking flask of culture medium, 37 DEG C, 5%CO2, 140rpm shake culture, primary every 2 days secondary cultures, inoculum density is
5×105Cells/ml.5 times or more secondary cultures are carried out continuously, when cell activity is restored to 95% or more, are cultivated suspending
Cell inoculation to expression culture medium in enter expression cultivation stage.
It expresses cultivation stage: pressing 5 × 105The density of cells/ml, the 5th seed cell more than generation is seeded to and is equipped with
In the 250mL shaking flask for expressing culture medium, volume of culture reaches 50mL, 33 DEG C, 5%CO2, 140rpm shake culture, continuously cultivate 11
It.Sampling 0.5mL daily measures the pH value of culture solution with micro pH meter, and measures cell with Vi-Cell XR cell counter
Density and Cell viability, culture middle and later periods detect concentration of glucose in culture solution with CEDEX Bio Biochemical Analyzer.When pH value is low
When 6.8, the 7.5%NaHCO of 1% (v/v) is added into culture shaking flask3Solution adjusts pH.When concentration of glucose is lower than 20mM
When, 45% glucose solution of 1% (v/v) is added into culture shaking flask.
When culture was to the 9th, 10,11 day, sampling 1mL takes supernatant after centrifugation, saves under the conditions of -20 DEG C, is used for purpose
Protein quantification detection.
When culture was to the 11st day, 5min will be centrifuged under the conditions of 800 × g of culture solution, supernatant is after 0.22 μm of filtration sterilization
Downstream is transferred to be purified.
(4) expression quantity detects: carrying out quantitative detection to destination protein using ELISA detection kit
(5) it purifies: the FSH in cell culture supernatant being purified using the method for affinity chromatography.What is used is affine
Chromatography media is the CaptureSelectTMFSH affine in immunity medium of Thermo company.Equalizing and buffering used in purification process
Liquid is 20mM phosphate buffer, and pH7.0-7.5, elution buffer is the 20mM phosphate buffer of the magnesium chloride containing 2M,
pH7.0-7.5.Purification process is to balance 5 column volumes of pillar, and loading, 3 column volumes of rebalancing elute 2 column volumes, is collected
Eluting peak.
(6) sialic acid content detects:
Sialic acid content detection is quantified using RP-HPLC method.Sample ultrafiltration after purification is changed into liquid into water, UV is fixed
The processing of sialic acid test sample is used for after amount.Respectively fetch water (as blank sample), sialic acid standard 1nmol, 3nmol,
5nmol, 7nmol and 20 μ g of test sample add 12 μ l of 2M sodium bisulfate with water by each sample polishing to 88 μ l, and concussion mixes,
80 DEG C of heating 20min are centrifuged 15s after cooling, 50 μ l of 20mg/ml hydrochloric acid o-phenylenediamine is added, and mix centrifugation 15s, again in 80 DEG C
Secondary heating 40min, after cooling, 450 μ l of mobile phase solution (600 μ l of total volume) is added in every pipe, mixes centrifugation, the inspection of HPLC loading
It surveys.Liquid phase chromatogram condition are as follows: chromatographic column Kromasil C18 chromatographic column (250 × 4.6mm, 5 μm)；Mobile phase: 0.15% butylamine,
0.5% phosphoric acid, 1.0% tetrahydrofuran, 2.0% acetonitrile；Flow velocity: 0.5ml/min, applied sample amount: 150 μ l, Detection wavelength: 230nm.
Detection finishes, and integrates to sialic acid chromatographic peak, is drawn and is marked to sialic acid content with the peak area of sialic acid standard solution
Directrix curve calculates sialic acid sum in test sample by the standard curve, counts sialic acid content.The calculating of sialic acid content is public
Formula are as follows: sialic acid content (mol/mol albumen)=(SA/P) * Mr
Wherein, SA is that sialic acid is total (nmol) in the test sample calculated according to standard curve；P is protein injection amount
(ng)；Mr is sample relative molecular mass.
The Chinese hamster ovary celI of embodiment 2-10, shaking flask culture expression Human Follicle Stimulating Hormone
Recovery expression Human Follicle Stimulating Hormone Chinese hamster ovary celI, according to embodiment 1 cultural method carry out secondary culture and
Expression culture, adds the NANA of various concentration after inoculation respectively, carries out destination protein expression quantity according to 1 the method for embodiment
Detection, purifying and sialic acid content detection, the results are shown in Table 1.
Table 1 adds influence of the NANA of various concentration to expressing quantity and sialic acid content
The results show that adding various concentration NANA meeting in the Chinese hamster ovary celI incubation of expression Human Follicle Stimulating Hormone
The expression quantity and sialic acid content of destination protein are had an impact.When NANA additive amount is greater than 20mM, the expression of destination protein
Amount is substantially reduced, and when NANA additive amount is less than 20mM, the expression quantity of destination protein is almost the same；When NANA additive amount is less than
When 5mM, with the raising of NANA concentration, sialic acid content has raised trend.When NANA additive amount is greater than 20mM, sialic acid
Content reduces.Comprehensively consider, while destination protein expression quantity maximum and sialic acid content can be made maximum, the addition concentration of NANA
For 5~10mM.More preferably 5mM.
The Chinese hamster ovary celI of embodiment 11, shaking flask culture expression recombinant human VEGF R-Fc fusion protein
The Chinese hamster ovary celI of 5mM NANA culture expression recombinant human VEGF R-Fc fusion protein is added in shake flask scale.
(1) culture medium and feed supplement
1. culture medium prepares: commercially available liquid Hycell CHO culture medium
2. feed supplement Excell Feed solution prepares: weighing the Excell Feed powder of 3.41g, ultrapure water dissolution and constant volume
To 100mL, then through 0.22 μm of filtration sterilization；
3.200mM L-Glutamine solution, 500mM NANA solution, 45% glucose solution, 7.5%NaHCO3Solution
Prepare with embodiment 1.
(2) instrument and equipment: with embodiment 1
(3) incubation:
Subculture stage: the L-Glutamine of 4mM is added first in Hycell CHO culture medium.
It recovers the Chinese hamster ovary celI for expressing recombinant human VEGF R-Fc fusion protein, is seeded to equipped with 20mL culture medium
In 125mL shaking flask, 37 DEG C, 5%CO2, 140rpm shake culture, it is primary every 2 days secondary cultures, inoculum density be 5 × 105Carefully
Born of the same parents/milliliter.5 times or more secondary cultures are carried out continuously, when cell activity is restored to 95% or more, expression culture can be seeded to
Stage.
It expresses cultivation stage: pressing 5 × 105The density of cells/ml, the 5th seed cell more than generation is seeded to and is equipped with
In the 500mL shaking flask of 100mL culture medium (94mL Hycell CHO culture medium, 6mL Excell Feed), volume of culture reaches
100mL, 34 DEG C, 5%CO2, 140rpm shake culture, continuously cultivate 13~15 days.It was respectively added at the 7th, 10,13 day respectively
The Excell Feed solution of 6mL.Sampling 0.5mL daily, with the pH value of micro pH meter measurement culture solution, and with Vi-Cell XR
Cell counter measures cell density and Cell viability, and the culture middle and later periods is detected in culture solution with CEDEX Bio Biochemical Analyzer
Concentration of glucose and expressing quantity.When pH value is lower than 6.9, the 7.5%NaHCO of 1% (v/v) is added into culture shaking flask3
Solution adjusts pH.When concentration of glucose is lower than 20mM, 45% glucose solution of 1% (v/v) is added into culture shaking flask.
After culture, 5min will be centrifuged under the conditions of 800 × g of culture solution, supernatant is transferred to after 0.22 μm of filtration sterilization
Downstream is purified.
Specific experiment operation:
2. 3. 1. take the shaking flask of three 500mL, 1. number is followed successively by；
2. 94mLHycell CHO culture medium (L-Glutamine containing 4mM) and 6mL Excell are added in three shaking flasks
Feed solution；
3. pressing 5 × 105The density of cells/ml is inoculated with；
4. the 500mM NANA solution that 1mL has been prepared 2. 3. is added in number shaking flask, 1. NANA direct sample is not added in shaking flask；
5. respectively sampling 0.5mL after mixing, cell density and motility rate are detected with Vi-cell XR cell counter；
6. being put into cell incubator culture, condition is 34 DEG C, 5%CO2,140rpm；
7. sampling 0.5mL to three shaking flasks daily, culture solution pH and cell density and motility rate are detected, and cultivating
Middle and later periods detects concentration of glucose and expressing quantity in culture solution with CEDEX Bio Biochemical Analyzer；
8. the 7.5%NaHCO of 1mL is added into culture shaking flask when pH value is lower than 6.93Solution adjusts pH.Work as grape
When sugared concentration is lower than 20mM, 45% glucose solution of 1mL is added into culture shaking flask；
9. culture was to the 15th day, harvested: culture solution is centrifuged 5min under the conditions of 800 × g, and supernatant is through 0.22 μm
Filtration sterilization saves under the conditions of 4 DEG C.
(4) it purifies:
The Fc fusion protein in cell culture supernatant is purified using the method for affinity chromatography.The affine layer used
Analyse the MabSelect SURE LX affinity media that medium is GE company.Equilibration buffer used in purification process is 20mM phosphorus
Phthalate buffer, pH7.0-7.5, elution buffer are citrate buffer containing 50mM, pH7.0-3.5.Purification process is flat
Weigh 5 column volumes of pillar, loading, and 3 column volumes of rebalancing elute 2 column volumes, collects eluting peak.With 1M Tris buffer
Destination protein is saved after eluent is neutralized to pH7.0-7.5.
(5) sialic acid content detects: carrying out sialic acid content detection according to 1 the method for embodiment.
(6) experimental result
The result is shown in Figure 1 and table 2.
2 sialic acid content of table
The results show that addition NANA generates the influence slightly inhibited to protein yield, but can in cell cultivation process
Effectively improve the sialic acid content of albumen.
The Chinese hamster ovary celI of embodiment 12,5L bioreactor culture expression Human Follicle Stimulating Hormone
(1) culture medium and feed supplement
1. expanding species culture medium prepares: commercially available liquid CDM4CHO culture medium
2.3.5w/v%Cell 6 solution of Boost prepares: weighing 6 powder of Cell Boost of 70g, ultrapure water dissolves simultaneously
It is settled to 2L, then through 0.22 μm of filtration sterilization；
3.200mM L-Glutamine solution prepares: weighing the L-Glutamine powder of 5.84g, ultrapure water dissolution and constant volume
To 200mL, then through 0.22 μm of filtration sterilization；(L-Glutamine mole is 146.14)
4.500mM NANA solution prepares: weighing the NANA powder of 7.73g, ultrapure water dissolves and be settled to 50mL, then
Through 0.22 μm of filtration sterilization；(NANA mole is 309.27)
5. expression culture medium prepares: measuring commercially available liquid Power CHO-2 culture medium 8L, be added what 200mL had been prepared
200mM L-Glutamine solution adds 6 solution of 3.5w/v%Cell Boost that 2L has been prepared, after mixing for use；
(whole process carries out in Biohazard Safety Equipment, guarantees sterile)
6.45% glucose solution prepares: weighing the glucose powder of 225g, ultrapure water dissolves and be settled to 500mL, so
By 0.22 μm of filtration sterilization；
7.7.5%NaHCO3Solution prepares: weighing the NaHCO of 37.5g3Powder, ultrapure water dissolve and are settled to 500mL, so
By 0.22 μm of filtration sterilization.
(2) instrument and equipment:
1.Thermo/371CO2Cell incubator
2.Infors/Celltron magnetic drives concussion shaking table
3. Su Jing is safe and sound/BSC-1300IIA2 Biohazard Safety Equipment
4.Roche/Cedex Bio biochemical process analyzer
The general water bath of 5.Vivo/VWB-9
The micro pH meter of 6.METTLER/S-210
310 5L bioreactor of 7.Eppendorf/Celligen
8.BECKMAN/Vi-Cell XR cell counter
9. Su Jing is safe and sound/SW-CJ-1F clean bench
(3) incubation:
Subculture stage: one cell of recovery is seeded in the 125mL shaking flask equipped with 20mL expanding species culture medium, 37 DEG C,
5%CO2, 140rpm shake culture, primary every 2 days secondary cultures, and carry out volume amplification, inoculum density is 5 × 105Cell/
Milliliter.5 secondary cultures are carried out continuously, when cell activity is restored to 95% or more, by the cell inoculation for the culture that suspends to table
Cultivation stage is expressed up to entering in culture medium.
Express cultivation stage: while two 5L bioreactors are run, the NANA of 0mM and 5mM is added respectively, is compared
Experiment, specific experiment operation:
1. 2. 1. two cleaning, assembling, sterilizing completely the same 5L reactors, number are respectively；
2. 5L expression culture medium is respectively added in two 5L reactors；
3. the NANA solution that 50mL has been prepared 2. is added in number bioreactor, 1. NANA direct sample is not added in shaking flask；
4. pressing 5 × 105The inoculum density of cells/ml is inoculated with two bioreactors respectively；
5. parameter setting is cultivated；(parameter specifically: stirring: 80rpm, temperature: 33 DEG C, dissolved oxygen: 30%, pH: >=
7.0)
6. respectively sampling 10mL after running 20min, with the pH value of micro pH meter detection culture solution, detected with cell counter
Cell density and motility rate；
7. sampling 10mL to two reactors daily, culture solution pH and cell density and motility rate are detected, and cultivating
Middle and later periods detects concentration of glucose in culture solution with Biochemical Analyzer；(when pH value is lower than 7.0, system will be automatically replenished 7.5%
NaHCO3Solution adjusts pH；When concentration of glucose is lower than 20mM, 45% glucose solution of 50mL is added into reactor)
8. culture was to the 9th, 10,11 day, two reactors respectively stay 10mL culture supernatant (after centrifugation), protect in -20 DEG C
It deposits；
9. culture was to the 11st day, culture solution is harvested through in-depth filtration (being followed by 0.22 μm of bacterial filter) system, so
After be handed over to downstream and purified.
(4) expression quantity detection, purifying, sialic acid content detection
The detection of destination protein expression quantity, purifying and sialic acid content detection are carried out according to 1 the method for embodiment.
(5) experimental result
As a result see Fig. 2 and table 3.
3 sialic acid content of table
The results show that 5mM NANA is added in cell cultivation process, with result one in shaking flask in 5L reactor scale
It causes, albumen sialic acid content can be effectively increased.
The Chinese hamster ovary celI of embodiment 13,5L bioreactor culture expression recombinant human VEGF R-Fc fusion protein
(1) culture medium and feed supplement
1. culture medium prepares: commercially available liquid Hycell CHO culture medium
2. feed supplement Excell Feed solution prepares: weighing the Excell Feed powder of 102.4g, ultrapure water dissolves and determines
Hold to 3L, then through 0.22 μm of filtration sterilization；
3.200mM L-Glutamine solution prepares: weighing the L-Glutamine powder of 5.84g, ultrapure water dissolution and constant volume
To 200mL, then through 0.22 μm of filtration sterilization；(L-Glutamine mole is 146.14)
4.500mM NANA solution prepares: weighing the NANA powder of 15.46g, ultrapure water dissolves and be settled to 100mL, so
By 0.22 μm of filtration sterilization；(NANA mole is 309.27)
5.45% glucose solution prepares: weighing the glucose powder of 450g, ultrapure water dissolves and is settled to 1L, then passes through
0.22 μm of filtration sterilization；
6.7.5%NaHCO3Solution prepares: weighing the NaHCO of 75g3Powder, ultrapure water dissolve and are settled to 1L, then pass through
0.22 μm of filtration sterilization.
(2) instrument and equipment: with embodiment 12
(3) incubation:
1. 2. 1. two cleaning, assembling, sterilizing completely the same 5L reactors, number are respectively；
2. 5L culture medium is respectively added in two 5L reactors (comprising 4600mL Hycell CHO culture medium, 100mL
200mM L-Glutamine solution, 300mL Excell Feed solution)；
3. the NANA solution that 50mL has been prepared 2. is added in number bioreactor, 1. NANA direct sample is not added in shaking flask；
4. pressing 5 × 105The inoculum density of cells/ml is inoculated with two bioreactors respectively；
5. parameter setting is cultivated；(parameter specifically: stirring: 80rpm, temperature: 34 DEG C, dissolved oxygen: 50%, pH:7.0
~7.2)
6. respectively sampling 10mL after running 20min, with the pH value of micro pH meter detection culture solution, detected with cell counter
Cell density and motility rate；
7. each to add 300mL Excell Feed solution when cultivating the 7th, 10,13 day；
8. sampling 10mL to two reactors daily, culture solution pH and cell density and motility rate are detected, and cultivating
Middle and later periods detects concentration of glucose and expressing quantity in culture solution with Biochemical Analyzer；(when pH value is higher than 7.2, system is certainly
It is dynamic to be passed through CO2It is adjusted；When being lower than 7.0, system is automatically replenished 7.5%NaHCO3Solution is adjusted；Work as concentration of glucose
When lower than 20mM, 45% glucose solution of 50mL is added into reactor)
9. culture was to the 15th day, culture solution is harvested through in-depth filtration (being followed by 0.22 μm of bacterial filter) system, so
After be handed over to downstream and purified.
(4) it purifies: being purified according to 11 the method for embodiment.
(5) sialic acid content detects: carrying out sialic acid content detection according to 1 the method for embodiment.
(6) experimental result
As a result see Fig. 3 and table 4.
4 sialic acid content of table
The results show that adding 5mM NANA in 5L reactor scale evaluation, in cell cultivation process to expressing quantity
And it is showed in the influence and shaking flask of albumen sialic acid content consistent.
The Chinese hamster ovary celI culture of embodiment 14,50L bioreactor culture expression Human Follicle Stimulating Hormone
(1) culture medium and feed supplement
1. expanding species culture medium prepares: commercially available liquid CDM4CHO culture medium
2. expression culture medium prepares: taking a 70L stainless steel liquid dispensing tank, the commercially available powder Power CHO-2 culture of 34L is added
Base (contains corresponding multicomponent), and 29.23g L-Glutamine powder is added, and 77.32g NANA powder is added, adds 350g
6 powder of Cell Boost, injection water dissolve and are settled to 45L, then through 0.22 μm of filtration sterilization；
3.45% glucose solution prepares: weighing the glucose powder of 675g, water for injection dissolves and be settled to 1.5L, so
By 0.22 μm of filtration sterilization；
4.7.5%NaHCO3Solution prepares: weighing the NaHCO of 112.5g3Powder, water for injection dissolve and are settled to 1.5L,
Then through 0.22 μm of filtration sterilization.
(2) instrument and equipment: with embodiment 12, wherein bioreactor is GE/XDR-50 50L bioreactor.
(3) incubation:
Subculture stage: one cell of recovery is seeded in the 125mL shaking flask equipped with 20mL expanding species culture medium, 37 DEG C,
5%CO2, 140rpm shake culture, primary every 2 days secondary cultures, and carry out volume amplification, inoculum density is 5 × 105Cell/
Milliliter.6 secondary cultures are carried out continuously, when cell activity is restored to 95% or more, by the cell inoculation for the culture that suspends to table
Cultivation stage is expressed up to entering in culture medium.
Expression cultivation stage: the culture process for adding 5mM NANA is amplified to 50L reactor pilot-scale, verifying technique
Can amplification.Specific experiment operation:
Then the installation of 1.50L disposable biological reaction bag fills 45L expression culture medium (NANA containing 5mM)；
2. parameter setting, stable operation；(parameter specifically: stirring: 80rpm, temperature: 33 DEG C, pH: >=7.0, ventilate:
0.1L Air/min)
3. the next day 5L seed cell is seeded in reactor sack, density reaches 5~7 × 10 after inoculation6Cell/milli
It rises；
4. it is 30% that dissolved oxygen control parameter, which is arranged,；
5. respectively sampling 10mL after running 20min, with the pH value of micro pH meter detection culture solution, detected with cell counter
Cell density and motility rate；
6. sampling 10mL daily detects culture solution pH and cell density and motility rate, and biochemical in the culture middle and later periods
Analyzer detects concentration of glucose in culture solution；(when pH value is lower than 7.0, system will be automatically replenished 7.5%NaHCO3Solution,
Adjust pH；When concentration of glucose is lower than 20mM, 45% glucose solution of 500mL is added into reactor)；
7. culture was to the 12nd day, culture solution is harvested through in-depth filtration (being followed by 0.22 μm of bacterial filter) system, so
After be handed over to downstream and purified.
(4) expression quantity detection, purifying, sialic acid content detection
The detection of destination protein expression quantity, purifying and sialic acid content detection are carried out according to 1 the method for embodiment.
(5) experimental result
As a result see Fig. 4 and table 5.
5 sialic acid content of table
| Incubation time d | Sialic acid content (mol/mol) |
| 8 | 9.46 |
| 9 | 10.02 |
| 10 | 10.68 |
| 11 | 11.01 |
| 12 | 10.93 |
The results show that adding 5mM NANA in cell cultivation process in 50L reactor scale, being reacted with shaking flask and 5L
The result of device is consistent, can effectively increase albumen sialic acid content.
The Chinese hamster ovary celI of embodiment 15,50L bioreactor culture expression recombinant human VEGF R-Fc fusion protein
(1) culture medium and feed supplement
1. culture medium prepares: taking a 70L stainless steel liquid dispensing tank, the commercially available powder Hycell CHO culture medium of 34L is added and (contains
Poloxamer 188, HEPES, NaHCO of corresponding amount3), 29.23g L-Glutamine powder is added, 77.32g NANA is added
Powder adds 102.4g Excell Feed powder, and injection water dissolves and be settled to 45L, then through 0.22 μm of filtration sterilization；
2. feed supplement Excell Feed solution prepares: weighing the Excell Feed powder of 341.3g, ultrapure water dissolves and determines
Hold to 10L, then through 0.22 μm of filtration sterilization；
3.45% glucose solution prepares: weighing the glucose powder of 900g, ultrapure water dissolves and is settled to 2L, then passes through
0.22 μm of filtration sterilization；
4.7.5%NaHCO3Solution prepares: weighing the NaHCO of 150g3Powder, ultrapure water dissolve and are settled to 2L, then pass through
0.22 μm of filtration sterilization.
(2) instrument and equipment: with embodiment 12, wherein bioreactor is GE/XDR-50 50L bioreactor.
(3) incubation:
Subculture stage: one cell of recovery is seeded in the 125mL shaking flask equipped with 20mL expanding species culture medium, 37 DEG C,
5%CO2, 140rpm shake culture, primary every 2 days secondary cultures, and carry out volume amplification, inoculum density is 5 × 105Cell/
Milliliter.6 secondary cultures are carried out continuously, when cell activity is restored to 95% or more, by the cell inoculation for the culture that suspends to table
Cultivation stage is expressed up to entering in culture medium.
Expression cultivation stage: the culture process for adding 5mM NANA is amplified to 50L reactor pilot-scale, verifying technique
Can amplification.Specific experiment operation:
Then the installation of 1.50L disposable biological reaction bag fills 45L culture medium (NANA containing 5mM)；
2. parameter setting, stable operation；(parameter specifically: stirring: 80rpm, temperature: 34 DEG C, pH:7.0~7.2, logical
Gas: 0.1L Air/min)
3. the next day 5L seed cell is seeded in reactor sack, density reaches 5~7 × 10 after inoculation6Cell/milli
It rises；
4. it is 50% that dissolved oxygen control parameter, which is arranged,；
5. respectively sampling 10mL after running 20min, with the pH value of micro pH meter detection culture solution, detected with cell counter
Cell density and motility rate；
6. each to add 300mL Excell Feed solution when cultivating the 6th, 9,12 day；
7. sampling 10mL daily detects culture solution pH and cell density and motility rate, and biochemical in the culture middle and later periods
Analyzer detects concentration of glucose and expressing quantity in culture solution；(when pH value is higher than 7.2, system is passed through CO automatically2It carries out
It adjusts；When being lower than 7.0, system is automatically replenished 7.5%NaHCO3Solution is adjusted；When concentration of glucose is lower than 20mM,
45% glucose solution of 500mL is added into reactor)
8. culture was to the 14th day, culture solution is harvested through in-depth filtration (being followed by 0.22 μm of bacterial filter) system, so
After be handed over to downstream and purified.
(4) it purifies: being purified according to 11 the method for embodiment.
(5) sialic acid content detects: carrying out sialic acid content detection according to 1 the method for embodiment.
(6) experimental result
As a result see Fig. 5 and table 6.
6 sialic acid content of table
| Incubation time d | Sialic acid content (mol/mol) |
| 8 | 8.22 |
| 9 | 8.75 |
| 10 | 8.95 |
| 11 | 8.34 |
| 12 | 8.47 |
| 13 | 8.45 |
| 14 | 8.32 |
The results show that adding 5mM NANA in cell cultivation process in 50L reactor scale, being reacted with shaking flask and 5L
The result of device is consistent, can effectively increase albumen sialic acid content.
Human Follicle Stimulating Hormone is expressed in embodiment 16-19, addition NeuGc ALPHA2-3Gal (NANG) shaking flask culture
Chinese hamster ovary celI
Recovery expression Human Follicle Stimulating Hormone Chinese hamster ovary celI, according to embodiment 1 cultural method carry out secondary culture and
Expression culture, adds the NeuGc ALPHA2-3Gal (NANG) of various concentration, according to 1 the method for embodiment after inoculation respectively
The detection of destination protein expression quantity, purifying and sialic acid content detection are carried out, the results are shown in Table 7.
Table 7 adds influence of the NANA of various concentration to expressing quantity and sialic acid content
The results show that adding various concentration NANG meeting in the Chinese hamster ovary celI incubation of expression Human Follicle Stimulating Hormone
The expression quantity and sialic acid content of destination protein are had an impact.Comprehensively consider, while destination protein expression quantity can be made maximum
With sialic acid content maximum, the addition concentration of NANA is 5~10mM.
Claims (10)
1. application of the sialic acid in the sialylation levels for improving expressing cho cell recombinant protein.
2. application according to claim 1, which is characterized in that the sialic acid is N-acetyl-neuraminate, N- hydroxyl acetyl
Neuraminic acid, deaminize at least one of neuraminic acid or neuraminic acid.
3. a kind of cultural method for the Chinese hamster ovary celI for expressing recombinant protein, which is characterized in that add saliva in cell cultivation process
Acid.
4. cultural method according to claim 3, which is characterized in that the concentration of the sialic acid is 5~10mM.
5. cultural method according to claim 3, which is characterized in that the sialic acid is N-acetyl-neuraminate, N- hydroxyl second
Acyl neuraminic acid, deaminize at least one of neuraminic acid or neuraminic acid；The recombinant protein is recombination human follicle stimulation
Element or recombinant human VEGF R-Fc fusion protein.
6. cultural method according to claim 3, which is characterized in that Chinese hamster ovary celI of the sialic acid in expression recombinant protein
It is inoculated in expression culture medium after secondary culture and carries out addition when expression culture.
7. cultural method according to claim 6, which is characterized in that the secondary culture is specially expression recombinant protein of recovering
Chinese hamster ovary celI, be seeded in expanding species culture medium, 37 DEG C, 5%CO2, 140rpm shake culture, it is primary every 2 days secondary cultures,
Inoculum density is 5 × 105Cells/ml is carried out continuously 5 times or more secondary cultures, when cell activity is restored to 95% or more,
The cell inoculation for the culture that suspends is subjected to expression culture into expression culture medium.
8. cultural method described according to claim 6 or 7, which is characterized in that the expression culture is specially by secondary culture
Seed cell is seeded in expression culture medium, adds sialic acid, 33 DEG C, 5%CO2, 140rpm shake culture, continuously cultivate 11-
15 days；Period controls pH value >=6.8 of culture solution, concentration of glucose >=20mM.
9. according to cultural method described in claim 3-8 any one, which is characterized in that further include culture solution after expression culture from
After the heart the step of supernatant liquid filtering, purifying.
10. cultural method according to claim 9, which is characterized in that the centrifugation is that 800 × g is centrifuged 5min；The filtering
For through 0.22 μm of membrane filtration；The purifying is affinitive layer purification.
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