CN113337562B

CN113337562B - Method for producing rhCG by using CHO (Chinese hamster ovary) cells through high-efficiency fermentation

Info

Publication number: CN113337562B
Application number: CN202110562698.6A
Authority: CN
Inventors: 夏星辉; 胡锦晶; 高乃波
Original assignee: Ningbo Renjian Pharmaceutical Group Co ltd
Current assignee: Ningbo Renjian Pharmaceutical Group Co ltd
Priority date: 2021-05-24
Filing date: 2021-05-24
Publication date: 2022-06-03
Anticipated expiration: 2041-05-24
Also published as: CN113337562A

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

Method for producing rhCG by using CHO (Chinese hamster ovary) cells through high-efficiency fermentation

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 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.

Further, the culture parameters were: 36.5 +/-1 ℃, pH value controlled at 6.8-7.2, dissolved oxygen controlled at 20-70 percent and rotation speed controlled at 60 +/-5 revolutions per minute.

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.

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 Tosoh;

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 fermentative 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):

when the volume is 20LCell density 1.5 x 10 in 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 fermentation HCG levels were determined using the kit described in example 1, and alpha and beta subunit transcript levels were determined by PCR using primers of 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 increase yield/reduce costs at similar yields, we have further improved the customized media 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 levels 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

1. A fed-batch culture fermentation method for expressing recombinant rhCG by using CHO cells, which is characterized in that the density is 1.5 x 10⁶Inoculating cells with the cell/ml or more into a fermentation mixed culture medium, determining the day after inoculation as the first day, and respectively adding a supplementary culture medium when culturing for 3 rd, 6 th and 9 th days; hydrocortisone at a concentration of 0.05mg/ml and sodium pyrophosphate at a concentration of 3mg/ml were included in the feed medium.

2. The method of claim 1, wherein the fermentation time is 12 days.

3. The method of claim 1, wherein the parameters during fermentation 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.