CN113337562A - Method for producing rhCG by using CHO (Chinese hamster ovary) cells through high-efficiency fermentation - Google Patents
Method for producing rhCG by using CHO (Chinese hamster ovary) cells through high-efficiency fermentation Download PDFInfo
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- CN113337562A CN113337562A CN202110562698.6A CN202110562698A CN113337562A CN 113337562 A CN113337562 A CN 113337562A CN 202110562698 A CN202110562698 A CN 202110562698A CN 113337562 A CN113337562 A CN 113337562A
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- 230000004151 fermentation Effects 0.000 title claims abstract description 34
- 101100468640 Danio rerio rhcgl2 gene Proteins 0.000 title claims abstract description 18
- 101150053759 rhcg gene Proteins 0.000 title claims abstract description 18
- 238000004519 manufacturing process Methods 0.000 title description 10
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- 239000001963 growth medium Substances 0.000 claims abstract description 31
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- 229940048086 sodium pyrophosphate Drugs 0.000 claims abstract description 20
- 235000019818 tetrasodium diphosphate Nutrition 0.000 claims abstract description 20
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 claims abstract description 20
- 229950000785 hydrocortisone phosphate Drugs 0.000 claims abstract description 16
- 229910021380 Manganese Chloride Inorganic materials 0.000 claims abstract description 14
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 claims abstract description 14
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims abstract description 14
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- 238000000034 method Methods 0.000 claims abstract description 14
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- OVBPIULPVIDEAO-UHFFFAOYSA-N N-Pteroyl-L-glutaminsaeure Natural products C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-UHFFFAOYSA-N 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
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- 239000001103 potassium chloride Substances 0.000 description 2
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- 238000011160 research Methods 0.000 description 2
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- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 2
- BBMHARZCALWXSL-UHFFFAOYSA-M sodium dihydrogenphosphate monohydrate Chemical compound O.[Na+].OP(O)([O-])=O BBMHARZCALWXSL-UHFFFAOYSA-M 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
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- 238000008157 ELISA kit Methods 0.000 description 1
- 208000007984 Female Infertility Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 206010021928 Infertility female Diseases 0.000 description 1
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 description 1
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- 229960002685 biotin Drugs 0.000 description 1
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- TWHXWYVOWJCXSI-UHFFFAOYSA-N phosphoric acid;hydrate Chemical compound O.OP(O)(O)=O TWHXWYVOWJCXSI-UHFFFAOYSA-N 0.000 description 1
- 230000003169 placental effect Effects 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
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- 231100000872 sexual dysfunction Toxicity 0.000 description 1
- 238000012807 shake-flask culturing Methods 0.000 description 1
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- 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 [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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- 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
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Abstract
The application provides a fed-batch culture fermentation method for expressing recombinant rhCG by using CHO cells, which comprises the steps of adding manganese chloride into a fermentation mixed culture medium, or adding hydrocortisone and sodium pyrophosphate into the fed-batch culture medium. The application also provides a corresponding culture medium for the method.
Description
Technical Field
The application belongs to the field of protein and the field of cell culture, and particularly provides a method for producing rhCG by using CHO cells through high-efficiency fermentation and a corresponding culture medium thereof.
Background
Human chorionic gonadotropin (hCG), a hormone synthesized by placental trophoblasts, consists of alpha and beta subunits, and is currently widely used for treating diseases such as female infertility, sexual dysfunction, abortion, dwarfism, skin itch, tumors and the like.
The hCG is prepared by taking urine of pregnant women as a raw material through methods such as precipitation, ion exchange chromatography and the like, contains more impurities, has uncertain sources, risks of infectious diseases, batch differences, easy occurrence of anaphylactic reaction and the like, and has risks in clinical use. Researchers have subsequently focused on the development of recombinant technology for production and have gradually replaced urinary HCG in the clinic.
Although there have been attempts to use hosts such as insect cells and nerve cells, the cell most commonly used in rhCG production is still a CHO cell. However, links such as an expression regulation mechanism of protein in CHO cells, a protein posttranslational modification regulation system, a cell growth metabolism regulation mechanism and the like are still lack of deep knowledge, so that theoretical guidance levels in aspects of exogenous gene integration strategies, screening of high-expression strains, efficient culture medium configuration, culture process regulation and the like are low, and related researches rely more on a large number of optimization screening and optimization experiments to obtain better operation conditions. The method has the advantages of low research and production efficiency, long time consumption, high cost and great influence by accidental factors. Although foreign advanced enterprises have developed Glutamine Synthetase (GS) systems, customized serum-free personalized media, optimized high-density microcarrier culture processes and feeding strategies, applied low-cost and low-shear bioreactors, high-level expression of rhCG still requires long culture times (> 20 days) and high production costs.
Therefore, there is still a need in the art for further improvements in the technology of CHO cell production of rhCG.
Disclosure of Invention
After factors such as fermentation parameters, cells and the like are optimized in the early stage, improvement of a fermentation mixed culture medium and a fed-batch culture medium is tried, the influence of various special additives on CHO expression rhCG is researched, and the fact that the expression level of the rhCG can be effectively improved by adding manganese ions, hydrocortisone and sodium pyrophosphate into the culture medium is found.
In one aspect, the present application provides a fed-batch fermentation method for expressing recombinant rhCG using CHO cells, comprising adding manganese chloride to a fermentation mixed medium, or adding hydrocortisone and sodium pyrophosphate to a feed medium.
Further, the concentration of manganese chloride added was 3mg/ml, and the concentrations of hydrocortisone and sodium pyrophosphate added were 0.05mg/ml and 3 mg/ml.
Further, the fermentation time was 12 days.
Further, the density was 1.5 x 106Inoculating cells of more than 20L/ml into 50L of mixed fermentation medium pre-cultured overnight, the day of inoculation is the first day, and the days of 3, 6 and 910L of feed medium was added separately.
Further, the culture parameters were: 36.5 +/-1 ℃, pH value controlled at 6.8-7.2, dissolved oxygen controlled at 20-70% and rotation speed controlled at 60 +/-5 r/min.
On the other hand, the application provides a culture medium for CHO cell expression recombination rhCG, which comprises a fermentation mixed culture medium and a feeding culture medium, wherein manganese chloride is added into the fermentation mixed culture medium, or hydrocortisone and sodium pyrophosphate are added into the feeding culture medium.
Further, the concentration of manganese chloride added was 3mg/ml, and the concentrations of hydrocortisone and sodium pyrophosphate added were 0.05mg/ml and 3 mg/ml.
In another aspect, the present application provides the use of manganese chloride, hydrocortisone, and/or sodium pyrophosphate in the preparation of a culture medium for recombinant rhCG expression in CHO cells.
Further, the concentration of manganese chloride in the medium was 3mg/ml, and the concentrations of hydrocortisone and sodium pyrophosphate were 0.05mg/ml and 3 mg/ml.
Drawings
FIG. 1 shows the basic process of producing rhCG by CHO cell fermentation.
FIG. 2 is a graph showing the relative abundance of each subunit of hCG at day 6 of fermentation culture.
Detailed Description
Example 1 cells, media and other reagents used for fermentation
The CHO cells used for the fermentation, made by the applicant, were transfected with vectors carrying rhCG alpha, beta subunits into CHO cell K1 strain using Lipofectamine TM2000 Lipofectase transfection reagent, which was subsequently acclimated without serum and had been used for more than 18 months in the applicant's pilot production of rhCG.
The applicant is a culture medium prepared in the fermentation process:
subculture medium:
CD CHO AGT 24.3mg/ml, dihydrogen phosphate monohydrate 2.69mg/ml, disodium hydrogen phosphate 4.33mg/ml, L-arginine 550mg/ml, L-asparagine 1300mg/ml, L-aspartic acid 400 mg/ml;
fermentation mixed culture medium:
24.3mg/ml of CD CHO AGT, 9.66mg/ml of CD OPTICHO AGT, 2.69mg/ml of monobasic sodium phosphate monohydrate, 4.33mg/ml of dibasic sodium phosphate, 0.15mg/ml of biotin, 11.5mg/ml of folic acid, 120mg/ml of inositol, 300mg/ml of potassium chloride, 5500mg/ml of glucose, 2100mg/ml of sodium chloride and 30mg/ml of soybean protein hydrolysate;
a supplemented medium:
balancd CHO Feee 366 mg/ml, sodium dihydrogen phosphate monohydrate 2.69mg/ml, disodium hydrogen phosphate 4.33mg/ml, L-arginine 2000mg/L, L-aspartic acid 4500mg/L, vitamin C15mg/L, vitamin H5 mg/L folic acid 30mg/L, inositol 400mg/L, vitamin B125 mg/L, potassium chloride 0.0015mg/L, F-68950 mg/L, glucose 18000 mg/L;
HCG ELISA kit: japanese east Cao;
MnCl2: jinan Kai Chuang chemical Co., Ltd;
CD CHO AGT：gibco
CD OPTICHO AGT：gibco
BalanCD CHO Feee3：irvine scientific
sodium pyrophosphate: food grade, Shandong Guante bioengineering, Inc.;
hydrocortisone: shanghai Michelin Biochemical technology, Inc.;
other reagents and instruments including PCR reagents and instruments are all made in the conventional countries.
EXAMPLE 2 basic fermentation production Process
Cell recovery:
1) and taking the cell freezing tube, and placing the tube in a water bath kettle at 37 ℃ until the frozen cell suspension is melted.
2) And (3) transferring the frozen cells to a centrifugal tube filled with 6-7 ml of CHO subculture medium, centrifuging at 1000rpm for 5 minutes, and removing the supernatant.
3) Gently blow and beat the conglomerated cells at the bottom in the centrifuge tube by using 10ml of CHO subculture medium sucked in times.
4) Inoculating into 125ml shake flask containing 10ml CHO subculture medium to obtain final volume of about 20ml, and inoculating with cell density of 0.4-1.0 x 106Individual cells/ml. Placing the shake flask in a carbon dioxide incubator at 36.5 + -1 deg.C and 5 + -3% CO2And 125. + -. 10 rpm.
Passage:
1) and (3) shake flask culture: placing the cell shake flask in a carbon dioxide incubator at 36.5 + -1 deg.C and 5 + -3% CO2And 125. + -. 10 rpm. Cell counts were performed daily when cell density reached 1.5 x 106When the cell density is above 0.4-1.0 x 10, adding CHO passage culture medium, and adjusting cell density6Individual cells/ml.
2) wave culture: when the cell density reaches 1.5 x 106Collecting cell sap in shake flask into sterile bottle, inoculating into cell culture bag via sterile tube connecting machine, and adjusting cell density to 0.4-1.0 × 106Each cell/ml, the cell culture bag was placed on a rocking apparatus at 36.5. + -. 1 ℃ and 20. + -. 5rpm for culture. When the cell density reaches 1.5 x 106When the cell density is above 0.4-1.0 x 10, adding CHO passage culture medium, and adjusting cell density6Individual cells/ml.
Fermentation: (100L scale):
cell density 1.5 x 10 in 20L volume cell culture bag6When the cell/ml is more than 20L, inoculating to 50L of fermentation mixed culture medium which is pre-cultured overnight, determining the first day on the day of inoculation, and adding 10L of feeding culture medium respectively on the 3 rd, 6 th and 9 th days of culture. Culturing for 12 days, and fermenting (culture parameters: 36.5 + -1 deg.C, pH 6.8-7.2, dissolved oxygen 40-60%, and rotation speed 60 + -5 rpm).
After and during the fermentation, HCG levels were measured using the kit described in example 1, and transcription levels of the alpha and beta subunits were measured by PCR using primers in the prior art (alpha subunit: upstream CTGGTCACATTGTGCTATCTTTC, downstream TGAGGTGACGTTCTTTTGGAC; beta subunit: upstream ATGTTCCAGGGGCTGCTGCTGTT, downstream CGCGGTAGTTATTGCACCACACCACCTGA)
Example 3 optimization of the fermentation Process
Through the optimization of factors such as fermentation parameters, cells and the like in the early stage, the expression level of the recombinant protein of about 180mg/L is realized within 12 days by the method. To further improve yield/at similar yieldThe amount is reduced and the cost is reduced, and we have made further improvements to the customized culture medium by themselves. With reference to the prior art, attempts were made to add MnCl to the culture medium2(3 mg/ml added to the fermentation mix), hydrocortisone, sodium pyrophosphate, the results are as follows:
TABLE 1 optimization of the effect of manganese ion addition to cell lines and fermentation Medium mixtures on expression levels (mean of three jar samples)
The results of further studies on the transcript level are shown in FIG. 2: we found that the ratio of beta subunit to alpha subunit in the optimized cell line significantly exceeded that of the original cell line, which means that alpha subunit transcriptional expression may be a yield-limiting factor in the optimized cell line, whereas MnCl was added to the fermentation mix2The transcription level of the alpha subunit can be effectively improved, and the yield is further improved;
TABLE 2 Effect of hydrocortisone and sodium pyrophosphate addition to feed medium on expression levels (optimized cell lines, three pot sample averages)
The addition of hydrocortisone or sodium pyrophosphate into the feed medium alone has no obvious effect on increasing the yield, while the combination of hydrocortisone and sodium pyrophosphate can realize the effect of obviously improving the expression amount of the recombinant protein (the primary molecule is that the combination of the two in the feed can inhibit the growth of cells and arrest the cell cycle to accumulate the product).
After combining manganese ions, hydrocortisone and sodium pyrophosphate, we achieved a high expression level of 280.22mg/L over a 12 day culture period.
Claims (9)
1. A fed-batch culture fermentation method for expressing recombinant rhCG by using CHO cells is characterized by comprising the step of adding manganese chloride into a fermentation mixed culture medium or adding hydrocortisone and sodium pyrophosphate into a feed culture medium.
2. The method of claim 1, wherein the concentration of manganese chloride added is 3mg/ml and the concentration of hydrocortisone and sodium pyrophosphate added is 0.05mg/ml and 3 mg/ml.
3. The method of claim 1 or 2, wherein the fermentation time is 12 days.
4. The method of claim 3, wherein the density is 1.5 x 106The cells with cells/ml above are inoculated into 50L of fermentation mixed culture medium which is pre-cultured overnight, the day of inoculation is the first day, and 10L of feed medium is added when the cells are cultured for 3 days, 6 days and 9 days.
5. The method according to any one of claims 1 to 4, wherein the culture parameters are: 36.5 +/-1 ℃, pH value controlled at 6.8-7.2, dissolved oxygen controlled at 20-70% and rotation speed controlled at 60 +/-5 r/min.
6. A culture medium for CHO cell expression recombination rhCG is characterized by comprising a fermentation mixed culture medium and a feeding culture medium, wherein manganese chloride is added into the fermentation mixed culture medium, or hydrocortisone and sodium pyrophosphate are added into the feeding culture medium.
7. A medium according to claim 6, wherein the manganese chloride is added at a concentration of 3mg/ml and the hydrocortisone and sodium pyrophosphate are added at a concentration of 0.05mg/ml and 3 mg/ml.
8. The application of manganese chloride, hydrocortisone and/or sodium pyrophosphate in preparing a culture medium for expressing recombinant rhCG by CHO cells.
9. Use according to claim 8, wherein the medium contains 3mg/ml manganese chloride and 0.05mg/ml hydrocortisone and 3mg/ml sodium pyrophosphate.
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