CN116731975A - Cell culture process for regulating acidic component of Fc fusion protein in Chinese hamster ovary cells and application of cell culture process - Google Patents
Cell culture process for regulating acidic component of Fc fusion protein in Chinese hamster ovary cells and application of cell culture process Download PDFInfo
- Publication number
- CN116731975A CN116731975A CN202310705897.7A CN202310705897A CN116731975A CN 116731975 A CN116731975 A CN 116731975A CN 202310705897 A CN202310705897 A CN 202310705897A CN 116731975 A CN116731975 A CN 116731975A
- Authority
- CN
- China
- Prior art keywords
- fusion protein
- cell culture
- culture
- acidic component
- culture process
- 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
- 108091006020 Fc-tagged proteins Proteins 0.000 title claims abstract description 48
- 230000002378 acidificating effect Effects 0.000 title claims abstract description 35
- 238000004113 cell culture Methods 0.000 title abstract description 30
- 210000004978 chinese hamster ovary cell Anatomy 0.000 title abstract description 9
- 230000001105 regulatory effect Effects 0.000 title abstract description 6
- 238000000034 method Methods 0.000 claims abstract description 23
- 239000012526 feed medium Substances 0.000 claims abstract description 13
- 238000004519 manufacturing process Methods 0.000 claims abstract description 5
- 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 14
- 239000008103 glucose Substances 0.000 claims description 14
- 238000012258 culturing Methods 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
- 239000001963 growth medium Substances 0.000 claims description 5
- 239000002609 medium Substances 0.000 claims description 5
- 239000007640 basal medium Substances 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 3
- 230000037452 priming Effects 0.000 claims 1
- 230000014616 translation Effects 0.000 claims 1
- 230000004048 modification Effects 0.000 abstract description 6
- 238000012986 modification Methods 0.000 abstract description 6
- 238000001816 cooling Methods 0.000 abstract description 5
- 230000008569 process Effects 0.000 abstract description 5
- 230000008901 benefit Effects 0.000 abstract description 3
- 230000004060 metabolic process Effects 0.000 abstract description 3
- 230000033228 biological regulation Effects 0.000 abstract description 2
- 230000015556 catabolic process Effects 0.000 abstract description 2
- 238000006731 degradation reaction Methods 0.000 abstract description 2
- 230000036961 partial effect Effects 0.000 abstract description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 28
- 210000004027 cell Anatomy 0.000 description 24
- 230000002829 reductive effect Effects 0.000 description 20
- 239000004310 lactic acid Substances 0.000 description 14
- 235000014655 lactic acid Nutrition 0.000 description 14
- 238000003306 harvesting Methods 0.000 description 13
- 239000000047 product Substances 0.000 description 10
- 235000018102 proteins Nutrition 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 6
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 6
- 230000001276 controlling effect Effects 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 6
- 230000003833 cell viability Effects 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 230000009467 reduction Effects 0.000 description 5
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 4
- 238000005277 cation exchange chromatography Methods 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 3
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000003513 alkali Substances 0.000 description 3
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 239000001569 carbon dioxide Substances 0.000 description 3
- 229910002092 carbon dioxide Inorganic materials 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 229910052708 sodium Inorganic materials 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 229940104230 thymidine Drugs 0.000 description 3
- 239000004472 Lysine Substances 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000009134 cell regulation Effects 0.000 description 2
- 230000006240 deamidation Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 238000006317 isomerization reaction Methods 0.000 description 2
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 2
- 230000000630 rising effect Effects 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 230000009450 sialylation Effects 0.000 description 2
- 229910000029 sodium carbonate Inorganic materials 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- LGZKGOGODCLQHG-CYBMUJFWSA-N 5-[(2r)-2-hydroxy-2-(3,4,5-trimethoxyphenyl)ethyl]-2-methoxyphenol Chemical compound C1=C(O)C(OC)=CC=C1C[C@@H](O)C1=CC(OC)=C(OC)C(OC)=C1 LGZKGOGODCLQHG-CYBMUJFWSA-N 0.000 description 1
- 241001075517 Abelmoschus Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 108010008165 Etanercept Proteins 0.000 description 1
- 102100026120 IgG receptor FcRn large subunit p51 Human genes 0.000 description 1
- 101710177940 IgG receptor FcRn large subunit p51 Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- NBBJYMSMWIIQGU-UHFFFAOYSA-N Propionic aldehyde Chemical compound CCC=O NBBJYMSMWIIQGU-UHFFFAOYSA-N 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 230000001174 ascending effect Effects 0.000 description 1
- 125000000613 asparagine group Chemical group N[C@@H](CC(N)=O)C(=O)* 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N aspartic acid group Chemical group N[C@@H](CC(=O)O)C(=O)O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- LGZKGOGODCLQHG-UHFFFAOYSA-N combretastatin Natural products C1=C(O)C(OC)=CC=C1CC(O)C1=CC(OC)=C(OC)C(OC)=C1 LGZKGOGODCLQHG-UHFFFAOYSA-N 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229960000403 etanercept Drugs 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 125000000404 glutamine group Chemical group N[C@@H](CCC(N)=O)C(=O)* 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229940125645 monoclonal antibody drug Drugs 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- SQVRNKJHWKZAKO-OQPLDHBCSA-N sialic acid Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@](O)(C(O)=O)OC1[C@H](O)[C@H](O)CO SQVRNKJHWKZAKO-OQPLDHBCSA-N 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- KZNICNPSHKQLFF-UHFFFAOYSA-N succinimide Chemical class O=C1CCC(=O)N1 KZNICNPSHKQLFF-UHFFFAOYSA-N 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- 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
-
- 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
- C12N2500/30—Organic components
- C12N2500/40—Nucleotides, nucleosides, bases
-
- 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
Abstract
The invention discloses a cell culture process for regulating an Fc fusion protein acidic component in Chinese hamster ovary cells and application thereof, and belongs to the field of cell culture. The cell culture process adopts a fed-batch culture mode for culture, and a first feed medium and a second feed medium are added at the same time from the cell culture to the 3 rd, 5 th, 7 th, 9 th and 11 th days. The method has the advantages that from the regulation and control of the metabolic process of cell culture, the modification and degradation of partial charge heterogeneity in the expression process are inhibited and prevented by changing the cooling time, the cooling temperature and the pH value, so that the acidic component level of the Fc fusion protein can be stably and effectively improved, the expression level of the Fc fusion protein can be simultaneously maintained or increased, the problems of charge heterogeneity and yield in the production of the Fc fusion protein can be effectively solved, and the method has wide adaptability and commercial application and good economic benefit.
Description
Technical Field
The invention relates to the field of cell culture, in particular to a cell culture process for regulating an Fc fusion protein acidic component in Chinese hamster ovary cells and application thereof.
Background
Mammalian expression systems are the systems that best achieve heterologous expression of recombinant proteins, the most representative and most widely used being chinese hamster ovary Cells (CHO). The Fc fusion proteins currently in the market and under investigation are mostly proteins expressed by CHO cells.
The Fc fusion protein is a novel protein produced by fusing an Fc segment of immunoglobulin with a certain functional protein molecule by utilizing a genetic engineering technology, and the functional protein molecule and the Fc segment can increase the stability of the fusion molecule, prolong the half life in vivo and reduce the immunogenicity of a medicament by increasing the molecular weight and an FcRn-mediated recycling mechanism after being fused, thereby improving the effectiveness, the safety, the medicament formation and the like of the protein medicament. Compared with the traditional monoclonal antibody drug (mAb), the Fc fusion protein has obvious clinical advantages in terms of patient preference and compliance, and has wider application. At present, about 20 Fc fusion protein medicines are marketed worldwide, and star products such as etanercept, abelmoschus, combretastatin belong to Fc fusion proteins.
During the production or storage of Fc fusion proteins, various enzymatic and chemical post-translational modifications may occur due to the complex culture environment, many of which may result in changes in the exposed charged residues on the surface of the protein or in changes in the acid dissociation constant, ultimately altering the overall charge distribution on the surface of the protein, which may result in the formation of charge heterosomes in the therapeutic protein. Proteins can be simply separated into acidic components, neutral main peaks and alkaline components according to their net charges. The most common charge heterogeneity modifications include: deamidation, isomerization, C-terminal lysine variation, N-terminal cyclization, sialylation, and the like. Generally, methylacetaldehyde of an arginine residue, deamidation, saccharification, sialylation, and trisulfide bond of an asparagine residue produce an acidic component; succinimides formed by isomerization of C-terminal lysine, N-terminal glutamine, C-terminal amidation and aspartic acid residues may produce basic components.
Some charge heterosomes, especially acidic charge heterosomes, may affect the immunogenicity, half-life, biological activity and stability of therapeutic Fc fusion proteins, and even may bring about unexpected side effects, thus affecting the safety and effectiveness of drugs, so that important attention and control are required in the process of Fc fusion protein drug development, and there are various means for controlling the acidic components of Fc fusion proteins during the development and cell culture stage, such as changing enzyme activity by changing the temperature of lowering, etc., so as to achieve the purpose of sialic acid modification and regulation, thus controlling the charge of the product, but many charge variation modifications occur in a molecule-dependent manner due to extracellular interactions, which are difficult to predict and control.
Therefore, how to optimize and regulate the acidic component to meet the quality requirement of the product through the cell culture process conditions is particularly critical, and how to improve the expression level of the Fc fusion protein and regulate the cell culture process of the acidic component becomes one of the difficult problems in the field.
Disclosure of Invention
The invention aims to provide a cell culture based on batch fed-batch culture, which can reduce the acid component proportion of Fc fusion protein and simultaneously improve the expression quantity of the Fc fusion protein, thereby obviously improving the quality and yield of the Fc fusion protein.
The first object of the present invention is to provide a method for increasing the expression level of an Fc fusion protein while reducing the acidic component, which comprises the following specific steps:
s1: inoculating the cells into a basic culture medium, controlling the temperature to be 36.5+/-0.5 ℃ and the pH value to be 7.00+/-0.20, and culturing for 0-2 days;
s2: and culturing until the 3 rd day, carrying out fed-batch culture, controlling the temperature to be 30.0+/-0.5 ℃, controlling the pH value to be 6.80+/-0.10, and continuing culturing for 10-12 days.
In one embodiment, the cells are seeded at a density of 0.8 to 1.2X10 6 cell/ml, stirring speed of 280rpm + -10 rpm, DO of 40% (. Gtoreq.20%),the air bottom ventilation is 10 (0-100) mL/min.
In one embodiment, the process feeds every 48 hours.
In one embodiment, the amount of each feed is 5% by weight of the initial culture of the first feed medium and 0.5% by weight of the initial culture of the second feed medium.
In one embodiment, the initial culture weight is 1.4 to 1.6kg.
In one embodiment, when the glucose concentration is below 3.00g/L, 300g/kg of glucose stock solution is used, with glucose being added to a final concentration of 5.00g/L.
In one embodiment, the basal medium comprises Eden B600S medium, 3-5 mM glutamine and 1% HT.
In one embodiment, the HT comprises a mixture of 10mM sodium hypoxanthine and 1.6mM thymidine.
In one embodiment, the first feed medium is Eden F600aS and the second feed medium is Eden F600bS.
The invention also provides application of the method in regulating and controlling the yield of Fc fusion protein and acidic components.
The invention also provides application of the method in producing Fc fusion proteins with low acidic components.
In one embodiment, the low acidity component refers to an acidity component of < 28.3%.
The beneficial effects are that:
(1) The cell culture process of the invention starts from the metabolic process regulation of cell culture, and obviously reduces the proportion of acidic components of Fc fusion protein by changing various conditions such as cooling time, cooling temperature, pH setting and the like in the cell culture process and using a commercial culture medium, wherein the content of acidic components is reduced from 25.3% to 17.0% and the main peak content is increased from 50.3% to 55.6%; the content of the acidic component is reduced from 28.3% to 20.6% on day 14, the main peak content is increased from 49.7% to 53.8%, the expression level of the Fc fusion protein is increased, and the yield is increased from 4.47g/L to 4.83g/L. It can be seen that the present invention provides a simple, easy to operate cell culture process that maintains the production process.
(2) The cell culture process provided by the invention has the effects of inhibiting and preventing the modification and degradation of partial charge heterogeneity in the expression process, so that the acidic component level of the Fc fusion protein can be stably and effectively improved, the expression quantity of the Fc fusion protein can be maintained or increased, and the problems of charge heterogeneity and yield in the production of the Fc fusion protein can be effectively solved.
Drawings
A plot of viable cell density versus culture time for the example of fig. 1;
FIG. 2 is a graph showing the change in cell viability over time in culture;
FIG. 3 is a graph showing the concentration of lactic acid as a function of time in culture;
FIG. 4 is a graph of glucose concentration versus incubation time for the example;
FIG. 5 is a graph showing comparison of the results of measurement of the expression level of Fc fusion proteins;
FIG. 6 is a graph comparing the results of detection of the acid component by cation Chromatography (CEX) in the example;
the results of the main peak detection by cation Chromatography (CEX) of the example of fig. 7 are compared.
Detailed Description
The invention starts from the metabolic process regulation of cell culture and optimizes by changing the temperature-reducing time, the temperature-reducing temperature and the pH value setting technological parameter conditions respectively. The invention is further illustrated by the following examples, which are not intended to limit the invention, but are merely illustrative thereof, in conjunction with the drawings and detailed description. Materials and the like used in the following examples are commercially available unless otherwise specified.
The main material source information in the following examples is shown in table 1 below:
seed culture medium: CD CHO AGT medium, 4mM glutamine, 1% HT (HT: 10mM sodium hypoxanthine and 1.6mM thymidine mixture).
Basal medium: eden B600S medium, 4mM glutamine, 1% HT (HT: 10mM sodium hypoxanthine and 1.6mM thymidine mixture).
TABLE 1 Main Material Source information Table
Example 1
The Fc fusion protein of example 1 was produced as follows:
cell resuscitation phase: and (3) taking out a CHO cell expressing the Fc fusion protein from the cell library, and rapidly thawing and recovering the cell in a water bath kettle at 37+/-0.5 ℃ for 120-180 seconds. Transferring the cell suspension into a preheated seed culture medium with the concentration of 25-35 mL, slightly mixing, sampling for cell counting, and regulating the cell inoculation density to 0.3X10 6 Placing the shaking bottle into a carbon dioxide shaking table for culturing for 2-4 days, wherein parameters of the shaking table are set as follows: rotational speed 130 rpm.+ -.10 rpm, CO 2 The concentration is 6.0% + -1.0%, and the temperature is 36.5+ -0.5 ℃.
Seed amplification stage: the number of living cells was measured as (0.5.+ -. 0.10). Times.10 with seed medium 6 The density of cells/ml was diluted to a new disposable sterile conical flask and placed on a carbon dioxide shaker: rotational speed 130 rpm.+ -.10 rpm, CO 2 The concentration is 6.0% +/-1.0%, the temperature is 36.5+/-0.5 ℃, the culture is carried out for 2-4 days, and the cells are gradually amplified until the number of living cells meets the requirement of the fed-batch seeding.
Cell fed-batch culture stage: the number of living cells cultured in the previous stage was determined to be (1.00.+ -. 0.20). Times.10 with the basal medium 6 cells/ml were inoculated into a 3L bioreactor at an initial culture weight of 1.5kg. Setting and monitoring various parameters: the temperature of the reactor is 36.5+/-0.5 ℃, the pH value is 7.00+/-0.20, DO is 40% (. Gtoreq.20%), the stirring speed is 280 rpm+/-10 rpm, and the gases (DO associated with oxygen and pH associated with air 10 (0-100) mL/min and carbon dioxide) are automatically controlled by the bioreactor. In the feeding process, 1mol/L sodium carbonate solution is associated with an alkali pump, the alkali pump and the pH, when the pH is lower than a set lower limit alkali pump, 1mol/L sodium carbonate solution is automatically fed, the cells are cultured until the 4 th day, and the culture temperature is reduced to 31.0+/-0.5 ℃. The cells were cultured until day 3, 5, 7, 9, and 11, respectively, fed with a first feed medium in an amount of 5% by weight of the initial culture and a second feed medium in an amount of 0.5% by weight of the initial cultureAnd (5) culturing. When the concentration of glucose was lower than 3.0g/L, 300g/kg of the glucose concentrate was added to the cell fluid to a concentration of 5.0g/L (containing 80.0g/kg of glucose in the first feed medium) every day, except for the day of harvest. Leave samples were cultured to day 12 and day 14, cultured to day 14 or when cell viability<The culture was terminated at 80%, and the supernatant was harvested.
The culture product was tested in this example, giving the following results: good cell growth state, and the highest viable cell density is 29.1X10 6 The cell viability is maintained above 98%, the glucose concentration is in the range of 2.8-6.5 g/L, the consumption is satisfied, the lactic acid shows the trend of rising and then falling, and the lactic acid concentration is 0.29g/L when harvesting; the expression level of the Fc fusion protein is 3.11g/L on day 12 and 4.47g/L at the time of harvest on day 14; the content of the acidic component is 25.3% at 12 days, and the main peak content is 50.3%; the acidic component content was 28.3% at day 14 and the main peak content was 49.7%.
Example 2
The cell culture process of this example is different from that of example 1 in that: the temperature reduction time of the cell culture in this example was from day 4, the temperature of the culture was reduced to 30.0deg.C.+ -. 0.5 ℃, and the rest of the procedures were the same as in example 1.
In this example, the culture product is tested by taking example 1 as a control group, as shown in fig. 1-7, compared with the control group, the cell growth condition of example 2 is better, the overall cell activity is not significantly different, the cell activity is more than 98% at harvest, the glucose level is maintained between 2.5g/L and 6.8g/L, consumption is satisfied, lactic acid shows a tendency of rising first and then falling, the difference in the lactic acid concentration is not significant at earlier stage, the lactic acid concentration in the culture product harvested in example 2 is higher after the 7 th day of culture, the lactic acid concentration at harvest is 0.67g/L, the Fc fusion protein expression level at the 12 th day of example 2 is improved to 3.16g/L, and the Fc fusion protein expression level at the 14 th day of harvest is improved to 4.52g/L; the content of the acidic component is reduced from 25.3% to 18.2% at 12 days, and the main peak content is reduced from 50.3% to 46.1%; the content of the acidic component is reduced from 28.3% to 19.0% on day 14, and the main peak content is reduced from 49.7% to 43.2%.
As is clear from the results of this example, decreasing the temperature of the decrease in temperature, increasing the yield of the Fc fusion protein, and also effectively decreasing the level of the acidic component, but decreasing the main peak content.
Example 3
The cell culture process of this example is different from that of example 1 in that: the temperature reduction time of the cell culture in this example was from day 3, the temperature of the culture was reduced to 31.0deg.C.+ -. 0.5 ℃, and the rest of the procedures were the same as in example 1.
In this example, the cultured product was tested by using example 1 as a control group, and as a result, referring to FIGS. 1 to 7, the living cell density was overall lower in example 3 than in the control group, the cell viability was maintained at 98% or more, and the glucose level was maintained at 3.1g/L to 6.8g/L, thereby satisfying the consumption. Lactic acid showed a tendency of ascending and descending, the difference in the early stage of lactic acid concentration was not significant, and the lactic acid concentration in the culture product harvested in example 3 was high after the culture was carried out until the 7 th day, and the lactic acid concentration at the time of harvesting was 0.88g/L. Example 3 reduction of Fc fusion protein expression to 3.01g/L on day 12 and reduction of Fc fusion protein expression to 4.14g/L at harvest on day 14; the content of the acidic component is reduced from 25.3% to 22.9% on day 12, and the main peak content is reduced from 50.3% to 49.3%; the content of the acidic component is reduced from 28.3% to 27.1% on day 14, and the main peak content is reduced from 49.7% to 47.4%.
As is clear from the results of this example, the content of the acidic component can be reduced by cooling in advance, but the main peak content is reduced, and the expression level of the Fc fusion protein is also significantly reduced.
Example 4
The cell culture process of this example is different from that of example 1 in that: the pH of the cell culture in this example was set to 6.90.+ -. 0.30, and the rest of the procedure was the same as in example 1.
In this example, the culture product was tested by using example 1 as a control group, and as a result, referring to fig. 1 to 7, the cell growth condition of example 4 was good, the whole was not significantly different, the cell viability was 98.5% at harvest, the lactic acid concentration was low, the lactic acid concentration was 0.19g/L at harvest, and the glucose level was maintained between 1.9g/L and 6.3g/L, so as to satisfy the consumption. Example 4 increase in Fc fusion protein expression to 3.29g/L on day 12 and increase in Fc fusion protein expression to 4.72g/L at harvest on day 14; the content of the acidic component rises from 25.3% to 26.1% on day 12, and the main peak content rises from 50.3% to 51.5%; the acidic component content was increased from 28.3% to 29.5% and the main peak content was increased from 49.7% to 50.8% on day 14.
From the results of this example, it was found that the yield of Fc fusion protein was significantly improved by optimizing pH setting, and the contents of acidic component and main peak were increased.
Example 5
The cell culture process of this example is different from that of example 1 in that: the temperature reduction time of the cell culture of this example was from day 4, the culture temperature was reduced to 30.0deg.C.+ -. 0.5 ℃, and the pH was changed to 6.80.+ -. 0.10.
In this example, the culture product was tested by using example 1 as a control group, and as a result, referring to fig. 1 to 7, the cell growth condition of example 5 was good, the whole was not significantly different, the cell activity was 98.9% at the time of harvesting, the lactic acid concentration was low, and the glucose level was maintained between 2.7g/L and 6.5g/L, so that the consumption was satisfied. Example 5 increase in Fc fusion protein expression to 3.31g/L on day 12 and increase in Fc fusion protein expression to 4.83g/L at harvest on day 14; the content of the acidic component is reduced from 25.3% to 17.0% on day 12, and the main peak content is increased from 50.3% to 55.6%; the content of the acidic component was reduced from 28.3% to 20.6% and the main peak content was increased from 49.7% to 53.8% on day 14.
From the results of this example, it was found that lowering the temperature to 30.0deg.C.+ -. 0.5deg.C and changing the pH to 6.80.+ -. 0.10 when the cell culture was carried out until day 4 effectively reduced the level of acidic components, increased the main peak content, and also significantly increased the Fc fusion protein expression to 4.83g/L.
From the results of examples 1 to 5 on days 12 and 14, it is understood that the increase in the culture period and the increase in the expression level of the Fc fusion protein, shortening the culture period, is advantageous in lowering the level of the acidic component and increasing the main peak content.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A method for increasing the expression level of an Fc fusion protein while reducing the acidic component, comprising the specific steps of:
s1: inoculating the cells into a basic culture medium, controlling the temperature to be 36.5+/-0.5 ℃ and the pH value to be 7.00+/-0.20, and culturing for 0-2 days;
s2: and culturing until the 3 rd day, carrying out fed-batch culture, controlling the temperature to be 30.0+/-0.5 ℃, controlling the pH value to be 6.80+/-0.10, and continuing culturing for 10-12 days.
2. The method according to claim 1, wherein the cells are seeded at a density of 0.8 to 1.2X10 6 The stirring speed of the cells/mL is 280rpm + -10 rpm, the DO is 40% (. Gtoreq.20%), and the air priming is 10 (0-100) mL/min.
3. The method according to claim 1 or 2, characterized in that the feeding is performed every 48 h.
4. A method according to claim 3, wherein the amount of each feed is 5% by weight of the initial culture of the first feed medium and 0.5% by weight of the initial culture of the second feed medium.
5. The method according to claim 4, wherein the initial culture weight is 1.4 to 1.6kg.
6. The method of claim 1, wherein glucose is added to a final concentration of 5.00g/L when the glucose concentration is less than 3.00 g/L.
7. The method of claim 1, wherein the basal medium comprises Eden B600S medium, 3-5 mM glutamine and 1% ht.
8. The method of claim 4, wherein the first feed medium is Eden F600aS and the second feed medium is Eden F600bS.
9. Use of the method of any one of claims 1 to 8 for modulating Fc fusion protein production and acidic components.
10. Use of the method according to any one of claims 1 to 8 for the production of Fc fusion proteins of low acidity component.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310705897.7A CN116731975A (en) | 2023-06-14 | 2023-06-14 | Cell culture process for regulating acidic component of Fc fusion protein in Chinese hamster ovary cells and application of cell culture process |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310705897.7A CN116731975A (en) | 2023-06-14 | 2023-06-14 | Cell culture process for regulating acidic component of Fc fusion protein in Chinese hamster ovary cells and application of cell culture process |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116731975A true CN116731975A (en) | 2023-09-12 |
Family
ID=87902382
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310705897.7A Pending CN116731975A (en) | 2023-06-14 | 2023-06-14 | Cell culture process for regulating acidic component of Fc fusion protein in Chinese hamster ovary cells and application of cell culture process |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116731975A (en) |
-
2023
- 2023-06-14 CN CN202310705897.7A patent/CN116731975A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20110189732A1 (en) | Process for the Fermentative Production of Erythropoietin | |
| JP5980772B2 (en) | Improved cell culture medium | |
| KR102358951B1 (en) | Mammalian cell culture | |
| KR102589415B1 (en) | Method and system for growing cell cultures while preventing lactate accumulation | |
| KR20100016271A (en) | Methods of protein production using anti-senescence compounds | |
| AU2011246502A1 (en) | Improved cell cultivation process | |
| KR19990072037A (en) | Method for producing recombinant protein of E. coli through cell high density fermentation | |
| MXPA03001487A (en) | Improved methods for growing mammalian cells in vitro. | |
| CN113502310B (en) | Method for preparing semaglutide precursor through high-density fermentation | |
| CN102382794B (en) | Perfusion culture method of mammal cell | |
| CN116731975A (en) | Cell culture process for regulating acidic component of Fc fusion protein in Chinese hamster ovary cells and application of cell culture process | |
| CN115521919B (en) | CHO cell culture method for regulating acidic charge isomers of PD-1 antibody and LAG-3 antibody | |
| CN113355286A (en) | Mammalian cell culture process for efficiently expressing recombinant feline interferon omega 2 | |
| US20130210075A1 (en) | Enhanced protein expression | |
| CN1267562C (en) | Production of peptides by fedbatch cultivation of microoganism | |
| EA022946B1 (en) | Process for obtaining aspart insulin using a pichia pastoris yeast strain | |
| KR20160113710A (en) | Perfusion media | |
| WO2022094418A1 (en) | Overexpression of insulin-like growth factor receptor mutants to modulate igf supplementation | |
| CN101061216A (en) | Methods for producing mammalian cells | |
| CN108823267B (en) | Method for regulating acid peak content of antibody secreted by CHO-K1 expression system | |
| CN115404222B (en) | CHO cell culture method | |
| CN115340984B (en) | CHO cell culture method | |
| EP2649176B1 (en) | Feed mixing device and its use | |
| CN114045235B (en) | Method for producing single-cell protein and fermentable sugar by using methanotrophic bacteria | |
| CN109517815B (en) | Novel method for improving expression quantity of saccharomyces cerevisiae cell surface display alpha-galactosidase prepared by fermentation tank |
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 |