CN113462648A - Mammalian cell culture process for efficiently expressing recombinant cat interferon omega 2 mutant - Google Patents
Mammalian cell culture process for efficiently expressing recombinant cat interferon omega 2 mutant Download PDFInfo
- Publication number
- CN113462648A CN113462648A CN202010241429.5A CN202010241429A CN113462648A CN 113462648 A CN113462648 A CN 113462648A CN 202010241429 A CN202010241429 A CN 202010241429A CN 113462648 A CN113462648 A CN 113462648A
- Authority
- CN
- China
- Prior art keywords
- culture
- serum
- medium
- cell
- supplementing
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 210000004962 mammalian cell Anatomy 0.000 title claims abstract description 15
- 101710169571 Interferon omega-2 Proteins 0.000 title claims abstract description 8
- 238000004113 cell culture Methods 0.000 title claims description 9
- 241000282326 Felis catus Species 0.000 title abstract description 12
- 210000004027 cell Anatomy 0.000 claims abstract description 35
- 230000001502 supplementing effect Effects 0.000 claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 13
- 230000008569 process Effects 0.000 claims abstract description 11
- 239000000413 hydrolysate Substances 0.000 claims abstract description 9
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 6
- 239000001301 oxygen Substances 0.000 claims abstract description 6
- 238000004114 suspension culture Methods 0.000 claims abstract description 3
- 239000002609 medium Substances 0.000 claims description 23
- 241000282324 Felis Species 0.000 claims description 10
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 8
- 239000008103 glucose Substances 0.000 claims description 8
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 5
- 239000007640 basal medium Substances 0.000 claims description 4
- 239000012526 feed medium Substances 0.000 claims description 4
- 239000001963 growth medium Substances 0.000 claims description 3
- 239000000654 additive Substances 0.000 claims description 2
- 230000003833 cell viability Effects 0.000 claims description 2
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims description 2
- 238000010899 nucleation Methods 0.000 claims description 2
- 230000000996 additive effect Effects 0.000 claims 1
- 230000014509 gene expression Effects 0.000 abstract description 17
- 108090000623 proteins and genes Proteins 0.000 abstract description 10
- 102000004169 proteins and genes Human genes 0.000 abstract description 6
- 230000000840 anti-viral effect Effects 0.000 abstract description 5
- 230000004083 survival effect Effects 0.000 abstract description 4
- 230000012010 growth Effects 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 102000008300 Mutant Proteins Human genes 0.000 description 14
- 108010021466 Mutant Proteins Proteins 0.000 description 14
- 108010050904 Interferons Proteins 0.000 description 10
- 102000014150 Interferons Human genes 0.000 description 9
- 229940079322 interferon Drugs 0.000 description 9
- 239000000047 product Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 5
- 241000701447 unidentified baculovirus Species 0.000 description 5
- 241000255789 Bombyx mori Species 0.000 description 3
- 208000035473 Communicable disease Diseases 0.000 description 3
- 241000238631 Hexapoda Species 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 108010045648 interferon omega 1 Proteins 0.000 description 3
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 2
- SHZGCJCMOBCMKK-DHVFOXMCSA-N L-fucopyranose Chemical compound C[C@@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O SHZGCJCMOBCMKK-DHVFOXMCSA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000013595 glycosylation Effects 0.000 description 2
- 238000006206 glycosylation reaction Methods 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000013595 supernatant sample Substances 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 1
- 241000680578 Canid alphaherpesvirus 1 Species 0.000 description 1
- 241000711506 Canine coronavirus Species 0.000 description 1
- 241000712083 Canine morbillivirus Species 0.000 description 1
- 241000701931 Canine parvovirus Species 0.000 description 1
- 241000288673 Chiroptera Species 0.000 description 1
- 238000011537 Coomassie blue staining Methods 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000714201 Feline calicivirus Species 0.000 description 1
- 241000713800 Feline immunodeficiency virus Species 0.000 description 1
- 241000714165 Feline leukemia virus Species 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 208000037319 Hepatitis infectious Diseases 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 241000711408 Murine respirovirus Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 241000711798 Rabies lyssavirus Species 0.000 description 1
- 206010051497 Rhinotracheitis Diseases 0.000 description 1
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000013475 authorization Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 208000005252 hepatitis A Diseases 0.000 description 1
- 230000009851 immunogenic response Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
Images
Classifications
-
- 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
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/34—Sugars
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
-
- 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
- C12N2510/00—Genetically modified cells
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Reproductive Health (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a mammalian cell suspension culture process for efficiently expressing recombinant cat interferon-omega 2 (rFeIFN-omega 2) mutants, which is carried out in a bioreactor and comprises the following steps: (1) setting the initial culture temperature to 36.5 ℃, dissolving oxygen at 40%, and reducing the temperature to 33 ℃ on the 6 th day of culture, wherein the pH is 7 +/-0.3; (2) supplementing 5%, 3% of serum-free supplemented medium 1 on days 3, 6, 8, 10, and 12, and feeding 2% of serum-free supplemented medium 2 on days 4, 7, 9, and 11; 6g/L of the hydrolysate was fed on days 5 and 8, respectively, dissolved oxygen was maintained at 40%, pH 7. + -. 0.3, and the culture was terminated on day 18. The invention has simple culture process flow, long survival rate of cells under high-density growth condition, high antiviral activity and low production cost, and the target protein expression can reach more than 2.5g/L, thereby being suitable for large-scale industrialized application.
Description
Technical Field
The invention relates to a mammalian cell culture process for efficiently expressing an rFeIFN-omega 2 mutant, belonging to the technical field of biological pharmacy.
Background
In 1985, interferon omega was first cloned from sendai virus-induced Namalwa cells and has now been identified as present in cats, pigs, horses, rabbits, bats, cattle and sheep. In 1992, the feline interferon gene was isolated by Nakamura, and then rFeiIFN-. omega.drugs were successfully developed and became the only approved formulation of feline interferon Omega, called Virbagen Omega, which has been widely used in Japan and European and American countries. The traditional Chinese medicine composition is mainly used for treating feline immunodeficiency virus, feline leukemia virus infectious diseases and canine parvovirus infectious diseases, and simultaneously has good treatment effect on infectious diseases such as feline viral rhinotracheitis, feline calicivirus, canine distemper virus, infectious hepatitis virus, rabies virus, pseudorabies virus, infectious gastroenteritis virus, canine coronavirus, canine herpes virus and the like.
In recent years, with the intensive research on Feline INTERFERON-omega, Chinese scholars Liuwen et al isolated 13 Novel Feline INTERFERONs-omega from cats in 2005, wherein-omega 2 obtained through a yeast expression system showed the strongest antiviral activity (Liuwen et al: Feline omega INTERFERON and its coding gene and application 2005, grant publication No. CN 1730491A) (Yang et al: Cloning and Characterization OF a Novel Feline IFN-omega. JOURNAL OF INTERFERON & CYTOKINE RESEARCH.2007; 27: 119-. In 2015, Huxiaoyuan and the like perform amino acid site-directed mutagenesis on cat omega 2 interferon by comparing 13 subtype gene sequences and amino acid sequences of cat omega interferon, clone genes encoding cat omega 2 interferon mutants into baculovirus transfer vectors to be recombined with baculovirus to infect insect hosts, express exogenous genes to obtain cat omega 2 interferon mutant proteins, and improve the antiviral activity of cat omega 2 interferon by more than 45% through mutagenesis (Huxiaoyuan and the like: cat omega 2 interferon mutants, a preparation method and application thereof 2015, an authorization notice number: CN 108276486A). If the cat omega 2 interferon mutant protein can be industrially produced, the method has good application prospect. Currently, FeINF-omega is successfully expressed mainly in escherichia coli, yeast and a silkworm baculovirus system, but rFeINF-omega expressed by escherichia coli is poor in activity, and core alpha 1,3 fucose with high proportion exists in an N-glycan structure of a product expressed by the silkworm baculovirus expression system, so that the product has potential immunogenic reaction (Minagawa S et al. novel recombinant viral expression N-polysaccharides with reduced produced by product of silkworm baculovirus expression system BMC Vet Res.2018; 14: 260).
Mammalian cell expression systems can direct the correct folding of proteins, providing multiple post-translational processing modification capabilities such as complex N-type glycosylation and accurate O-type glycosylation, and thus the expression products are closest to natural higher biological protein molecules in terms of molecular structure, physicochemical properties and biological functions. The feline belongs to mammals, and rFeINF-omega is a glycoprotein, and a foreign protein produced by post-translational modification of mammalian cells is higher than a prokaryotic expression system and eukaryotic expression systems such as yeast and insect cells in terms of activity, is closer to a natural protein, and can overcome the immunogenicity risk caused by N-glycan chain core alpha 1-3 fucose peculiar to an insect or plant cell expression system. However, mammalian cell expression systems are costly and inefficient. Therefore, the invention develops a process method which is simple and easy to control and low in cost, the yield of the rFeINF-omega 2 mutant protein produced by the process can reach more than 2.5g/L, and the antiviral activity of the product is obviously superior to that of other expression systems.
Disclosure of Invention
The invention aims to provide a mammalian cell culture process for efficiently expressing an rFeINF-omega 2 mutant, which solves the problems of low efficiency and high cost of mammalian cell expression. Specifically, a production culture process with simple and easy control operation, low cost, high expression product yield and high yield is provided for the industrialization of the rFeINF-omega 2 mutant protein, and the main technical scheme is as follows:
inoculating the mammalian cells expressing the rFeINF-omega 2 mutant into a bioreactor containing a serum-free basal medium for suspension culture, controlling the temperature, dissolved oxygen, pH and rotating speed in the culture process, feeding different serum-free supplemented media, hydrolysate and glutamine regularly in the culture process, monitoring the growth condition of the cells and supplementing a glucose solution.
Preferably, the mammalian cell is a chinese hamster ovary cell, preferably a CHO DG44 cell.
Preferably, the cell seeding density is 5-8X 105Per mL, preferably 5X 105/mL。
Preferably, the working volume of the basic culture medium of the bioreactor is 1-500L, the rotating speed is set to be 10-200 r/min, and specifically,
the working volume of the medium in a 7 liter bioreactor is 1-5L, preferably 3L; setting the rotation speed of 100 and 200 revolutions per minute, preferably 150 revolutions per minute;
the working volume of the medium in a 50 liter bioreactor is 10-40L, preferably 30L; setting the rotating speed to be 80-120 revolutions per minute, preferably 90 revolutions per minute;
the working volume of the medium in the 250 liter bioreactor is 40-200L, preferably 150L; setting the rotating speed to be 10-80 revolutions per minute, preferably 50 revolutions per minute;
preferably, the temperature is controlled by setting the initial culture temperature to be 36-38 ℃, preferably 36.5 ℃; the culture temperature was decreased to 33 ℃ from the 6 th day of the culture.
Preferably, the dissolved oxygen control condition is 30% -80%, preferably 40%;
preferably, the pH control range is 5-7.5, preferably 7 +/-0.3;
preferably, the feeding mode of the serum-free feeding medium 1 is that 5%, 3% of the serum-free feeding medium is respectively fed on days 3, 6, 8, 10 and 12; the feeding mode of the serum-free feed medium 2 is that 2 percent of the serum-free feed medium is respectively fed on days 4, 7, 9 and 11;
preferably, the hydrolysis material is fed in a mode of feeding 6g/L of hydrolysis material on days 5 and 8 respectively;
preferably, the serum-free basal medium consists of one or more of ActiPro medium with the concentration of 10-30g/L and SFM4CHO medium with the concentration of 10-20 g/L; and additives, which are composed of one or more of F68 with the concentration of 0.5-2g/L, glucose with the concentration of 3-5g/L and glutamine with the concentration of 4-8mM and the pH value of 7-7.5.
Preferably, the serum-free supplemented medium 1 consists of one or more of Cell Boost 7a with the concentration of 100-200 g/L and Cell Boost 7b supplemented with the concentration of 50-100 g/L; the serum-free supplemented medium 2 consists of EX-CELL Advanced CHO Feed 1 with the concentration of 50-100 g/L.
Preferably, the hydrolysate is composed of one or more of Hypep 7504 with the concentration of 50-200 g/L and PF Plus ACF hydrolysate with the concentration of 50-200 g/L;
the invention has the beneficial effects that: provides a simple and high-efficiency mammalian cell culture process for expressing the rFeINF-omega mutant, so that the expression product has high yield, low cost and good antiviral activity, and is suitable for large-scale industrial application.
Drawings
FIG. 1 shows the curves of the number of days and cell density of rFeINF- ω mutant protein-expressing cells cultured in a bioreactor
FIG. 2 shows the curves of the number of days of culture and the cell viability of rFeINF-omega mutant protein-expressing cells in a bioreactor
FIG. 3 shows the SDS-PAGE examination result of culture supernatant samples of rFeINF-omega mutant protein-expressing cells in a bioreactor (reduction)
FIG. 4 shows the expression level of rFeINF-omega mutant protein expressing cells in culture supernatant samples in a bioreactor
Detailed Description
The following examples are provided to further understand and explain the present invention and are not to be construed as limiting the invention.
Example 1 recombinant Cat interferon-omega 2 mutant protein expressing cell expansion culture
Resuscitating mammalian cells expressing recombinant feline interferon-omega 2 mutant at 5X 105Inoculating cells/ml density into a cell shake flask, placing the cell shake flask into a shaking table for shaking culture, and setting parameters of the shaking table as follows: 36.5 ℃ and 5% CO2140 rpm/min; after continuous culture for about 3 days, the cell density reaches 2-3X 106cells/ml at 5X 105Subculturing is carried out at a cell/ml density until the number of cells is sufficient to inoculate the bioreactor, and seed cell liquid is obtained.
Example 2 recombinant Cat interferon-omega 2 mutant protein expressing cell bioreactor culture
Mixing seed cell liquid at 5 × 105cells/ml are inoculated into a bioreactor (the working volume is 3L), an ActiPro culture medium is adopted for culture, and the set technological parameters are as follows: culturing at 36.5 deg.C, dissolved oxygen content of 40%, pH of 7 + -0.3, and stirring speed of 150 rpmDay 6, the temperature was reduced to 33 ℃; feeding 5%, 3% Cell Boost 7a/Cell Boost 7b (10:1 ratio) on days 3, 6, 8, 10, 12 respectively, feeding 2% EX-CELL Advanced CHO Feed 1 on days 4, 7, 9, 11 respectively, feeding 6g/L of Hypep 7504 hydrolysate on days 5 and 8 respectively, and feeding 1% 200mM glutamine on day 3; taking cell supernatant every day to perform biochemical index measurement, and supplementing 1% 400g/L glucose solution when the glucose concentration is lower than 2g/L to keep the glucose concentration above 2 g/L. Cells were cultured continuously until the peak density reached 3.27X 10 at day 87cells/ml (figure 1), the survival rate is maintained at a constant level after the culture under the high-density growth condition for 14 days, the survival rate is slightly reduced after the culture for 15 days, and the cell survival rate is maintained above 80 percent after the culture for 18 days (figure 2), and the culture is stopped to obtain cell stock solution.
Example 3 yield assessment of recombinant feline interferon-omega 2 mutant proteins
Supernatant samples from 12 th, 14 th, 16 th and 18 th days of culture in the bioreactor were subjected to SDS-PAGE and Coomassie blue staining to identify that a clear rFeIFN-omega 2 mutant protein band existed at about 20kDa and the expression level of the rFeIFN-omega 2 mutant protein increased with the increase of culture time (FIG. 3). While the supernatant samples were purified by affinity chromatography and then the purified rfefn- ω 2 mutant proteins were quantified by BCA, the results also showed elevated protein expression levels with increasing culture time, up to 2.61g/L at day 18 of harvest (figure 4). Another evaluation of the specific activity of the purified product against VSV virus showed that the biological specific activity of the rFeIFN-omega 2 mutein produced by the inventive culture process against VSV>3×108IU/mg。
Claims (5)
1. A mammalian cell culture process for efficiently expressing a recombinant feline interferon-omega 2 (rFeIFN-omega 2) mutant is characterized by comprising the following specific steps:
(1) inoculating the conventionally prepared seed cells into a bioreactor containing a serum-free basal medium for suspension culture;
(2) the cell seeding density is 5-8 × 105cellsPer mL, preferably 5X 105cells/mL;
(3) The working volume of the basic culture medium in the bioreactor is 1-500L, the rotating speed is set to be 10-200 r/min, and specifically,
the working volume of the medium in a 7 liter bioreactor is 1-5L, preferably 3L; setting the rotation speed of 100 and 200 revolutions per minute, preferably 150 revolutions per minute;
the working volume of the medium in a 50 liter bioreactor is 10-40L, preferably 30L; setting the rotating speed to be 80-120 revolutions per minute, preferably 90 revolutions per minute;
the working volume of the medium in the 250 liter bioreactor is 40-200L, preferably 150L; setting the rotating speed to be 10-80 revolutions per minute, preferably 50 revolutions per minute;
(4) setting the initial culture cell temperature at 36-38 deg.c, preferably 36.5 deg.c;
(5) setting the dissolved oxygen condition at 30-80%, preferably 40%;
(6) controlling the pH value to be 5-7.5, preferably 7 +/-0.3;
(7) supplementing 3-10% of serum-free supplemented medium 1, preferably 5% on the 3 rd day of culture, and simultaneously supplementing 1-5 mM of glutamine, preferably 2 mM; supplementing glucose solution in the culture process to ensure that the content of glucose in the culture solution is not lower than 2g/L, and controlling the cell viability to be more than 70% in the culture process;
(8) supplementing 1-5% of serum-free supplemented medium 2, preferably 2% on the 4 th day of culture;
(9) supplementing 1-10 g/L of hydrolysate on the 5 th day of culture, preferably 6 g/L;
(10) supplementing 5% of serum-free supplemented medium 1 again on the 6 th day of culture, and adjusting the temperature to 33 ℃ for continuous culture;
(11) supplementing 2% of serum-free supplemented medium 2 again at the 7 th day of culture, and maintaining the temperature at 33 ℃ for continuous culture;
(12) supplementing 5% serum-free supplemented medium 1 and 6g/L hydrolysate again at 8 days of culture, and maintaining the temperature at 33 deg.C for further culture;
(13) supplementing 2% of serum-free supplemented medium 2 again at the 9 th day of culture, and maintaining the temperature at 33 ℃ for continuous culture;
(14) supplementing 5% of serum-free supplemented medium 1 again at 10 days of culture, and maintaining the temperature at 33 ℃ for continuous culture;
(15) supplementing 2% of serum-free supplemented medium 2 again on the 11 th day of culture, and continuing the culture at the temperature of 33 ℃;
(16) the culture was continued at 33 ℃ by supplementing the culture with 3% of serum-free feed medium 1 again on day 12.
2. The process of claim 1, wherein the mammalian cell is a Chinese hamster ovary cell, preferably a CHO DG44 cell.
3. The cell culture process according to claim 1, wherein the serum-free basal medium consists of one or more of ActiPro medium with concentration of 10-30g/L and SFM4CHO medium with concentration of 10-20 g/L; the additive is composed of one or more of F68 with concentration of 0.5-2g/L, glucose with concentration of 3-5g/L, and glutamine with concentration of 4-8mM, and has pH value of 7-7.5.
4. The Cell culture process according to claim 1, wherein the serum-free feed medium 1 comprises one or more of Cell Boost 7a at a concentration of 100-200 g/L and Cell Boost 7b at a concentration of 50-100 g/L; the serum-free supplemented medium 2 consists of EX-CELL Advanced CHO Feed 1 with the concentration of 50-100 g/L.
5. The cell culture process according to claim 1, wherein the hydrolysate is composed of one or more of Hypep 7504 with a concentration of 50-200 g/L and PF Plus ACF hydrolysate with a concentration of 50-200 g/L.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010241429.5A CN113462648A (en) | 2020-03-31 | 2020-03-31 | Mammalian cell culture process for efficiently expressing recombinant cat interferon omega 2 mutant |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202010241429.5A CN113462648A (en) | 2020-03-31 | 2020-03-31 | Mammalian cell culture process for efficiently expressing recombinant cat interferon omega 2 mutant |
Publications (1)
Publication Number | Publication Date |
---|---|
CN113462648A true CN113462648A (en) | 2021-10-01 |
Family
ID=77865257
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202010241429.5A Pending CN113462648A (en) | 2020-03-31 | 2020-03-31 | Mammalian cell culture process for efficiently expressing recombinant cat interferon omega 2 mutant |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113462648A (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113355286A (en) * | 2020-03-02 | 2021-09-07 | 成都彤琦恩生物科技有限公司 | Mammalian cell culture process for efficiently expressing recombinant feline interferon omega 2 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105462909A (en) * | 2015-12-31 | 2016-04-06 | 哈药集团技术中心 | Culture method for high-efficiency human follicle stimulating hormone expression CHO cells |
CN107460159A (en) * | 2017-08-14 | 2017-12-12 | 上海多宁生物科技有限公司 | Serum-free, without albumen supplemented medium and preparation method thereof and use |
CN107760651A (en) * | 2016-08-23 | 2018-03-06 | 四川科伦博泰生物医药股份有限公司 | A kind of cell culture medium and production method of protein |
CN108251375A (en) * | 2016-12-28 | 2018-07-06 | 康立泰药业有限公司 | A kind of method of fermenting and producing rhIL-12 |
CN108276486A (en) * | 2015-04-23 | 2018-07-13 | 中国农业科学院生物技术研究所 | 2 interferon mutants of cat ω and its preparation method and application |
CN109055426A (en) * | 2018-08-06 | 2018-12-21 | 苏州迈缔姆生物技术有限公司 | A method of Expression product class people lubricates element in Chinese hamster ovary cell |
-
2020
- 2020-03-31 CN CN202010241429.5A patent/CN113462648A/en active Pending
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108276486A (en) * | 2015-04-23 | 2018-07-13 | 中国农业科学院生物技术研究所 | 2 interferon mutants of cat ω and its preparation method and application |
CN105462909A (en) * | 2015-12-31 | 2016-04-06 | 哈药集团技术中心 | Culture method for high-efficiency human follicle stimulating hormone expression CHO cells |
CN107760651A (en) * | 2016-08-23 | 2018-03-06 | 四川科伦博泰生物医药股份有限公司 | A kind of cell culture medium and production method of protein |
CN108251375A (en) * | 2016-12-28 | 2018-07-06 | 康立泰药业有限公司 | A kind of method of fermenting and producing rhIL-12 |
CN107460159A (en) * | 2017-08-14 | 2017-12-12 | 上海多宁生物科技有限公司 | Serum-free, without albumen supplemented medium and preparation method thereof and use |
CN109055426A (en) * | 2018-08-06 | 2018-12-21 | 苏州迈缔姆生物技术有限公司 | A method of Expression product class people lubricates element in Chinese hamster ovary cell |
Non-Patent Citations (1)
Title |
---|
RAMANI WONDERLING 等: "Cloning, expression, purification, and biological activity of five feline type I interferons", VETERINARY IMMUNOLOGY AND IMMUNOPATHOLOGY, pages 21 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113355286A (en) * | 2020-03-02 | 2021-09-07 | 成都彤琦恩生物科技有限公司 | Mammalian cell culture process for efficiently expressing recombinant feline interferon omega 2 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP2564506B2 (en) | Method for producing heterologous protein | |
KR101828624B1 (en) | Improved cell cultivation process | |
CN1187441C (en) | Media for culturing animal cells and process for producing protein by using same | |
CN1391604A (en) | Medium for protein-free and serum-free cultivation of cells | |
US4656131A (en) | Method for producing interferons | |
CN111454877B (en) | CHO cell culture method | |
EP1527188B1 (en) | Process for the production of increased amounts of correctly folded heterologous protein in inclusion bodies | |
CN113355286A (en) | Mammalian cell culture process for efficiently expressing recombinant feline interferon omega 2 | |
CN113462648A (en) | Mammalian cell culture process for efficiently expressing recombinant cat interferon omega 2 mutant | |
Yim et al. | High-level secretory production of human granulocyte-colony stimulating factor by fed-batch culture of recombinant Escherichia coli | |
ZA200101394B (en) | Production of human erythropoietin. | |
JP5460660B2 (en) | Method for adjusting human G-CSF | |
RU2575598C9 (en) | Method for obtaining alpha5-interferon protein | |
JP2977241B2 (en) | Optimized fermentation method for exogenous protein production in Escherichia coli | |
CN112391431B (en) | Fermentation medium and fermentation method for recombinant leukocyte inhibitory factor and hirudin chimeric protein | |
RU2680886C1 (en) | RECOMBINANT PLASMID pFM-GCSF5 THAT PROVIDES EXPRESSION OF SYNTHETIC HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR, ESCHERICHIA COLI FM-GCSF BACTERIA STRAIN - PRODUCER OF RECOMBINANT HUMAN GRANULOCYTE COLONY-STIMULATING FACTOR AND METHOD FOR PRODUCTION THEREOF | |
CN115340984B (en) | CHO cell culture method | |
CN109055464B (en) | Culture medium for producing recombinant human brain natriuretic peptide and fermentation process | |
EA013263B1 (en) | Process for preparing interferon beta | |
CN115287245A (en) | High-density fermentation process of secretory recombinant human growth hormone engineering bacteria | |
CN113969257A (en) | Culture medium for producing insulin glargine | |
CN118126912A (en) | Fermentation medium of interleukin 6 and fermentation method thereof | |
CN117384989A (en) | Quality control method for reducing acid charge heteroplastid level of antibody protein and application thereof | |
CN85102303A (en) | Produce the method for interferon | |
CN115404222A (en) | CHO cell culture method |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20211001 |
|
WD01 | Invention patent application deemed withdrawn after publication |