CHO (Chinese hamster ovary) cell and suspension process method

Abstract

The invention discloses a CHO (Chinese hamster ovary) cell and a suspension process method, which are characterized in that a stable high-expression cell strain is obtained by improving a target expression vector, and the protein expression quantity of 1g / L is realized. The improved CHO cell can be suitable for a WAVE wave bioreactor and serum-free culture, contamination is effectively prevented, interference of exogenous factors is reduced, the level of related impurities such as endotoxin in a cell expression product is reduced, the operation steps are simplified, the occupied culture space is reduced, meanwhile, the culture time is shortened, and the culture efficiency is improved. Therefore, the culture efficiency is improved, bacterial contamination is effectively prevented, the endotoxin level in the product is reduced, and the production cycle is greatly shortened.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine production, and in particular to a CHO cell and a suspension process method. Background Art

[0002] Tenecteplase for injection cannot be separated from CHO cell culture technology in the production process. There are currently two main commonly used CHO cell culture processes: one is to carry out small-scale, manual cell culture in a culture dish by adherence, such as the PE-coated plastic culture dish commonly seen on the market from Corning, which has the characteristics of low production cost, simple operation, and stable cell growth state; the other is a cell factory that can carry out large-scale amplification of adherent cells, which has been developed in recent years based on the above technology and is also widely used in research and application fields such as vaccine production and antibody preparation; however, in actual production, both of the above cell culture methods have certain shortcomings, among which the shortcomings of low space utilization of culture dishes and low manual operation efficiency make them unable to be applied on a large scale, and the cells in the cell factory are difficult to observe and adjust, which leads to uneven cell distribution during their growth process, and the cell yield per unit area is significantly lower than that of the culture dish, and there is a risk of exogenous viruses such as serum in adherent culture, and quality differences between batches affect product quality.

[0003] The traditional adherent culture process uses serum during cell expansion, which is not in line with the current industry development trend. The quality differences between serum batches, the risk of exogenous viruses, and the risk of TSE / BSE transmission will all affect the safety and effectiveness of the product. At present, the commercial production scale of biological products is large. Once the system is contaminated, it will bring large raw material costs and time costs. Generally, the international standard contamination rate is less than 1%. Therefore, the design principle of the production line is the simpler the better, and the fewer exposed links the better.

[0004] Therefore, it is necessary to provide a new technical solution to overcome the above-mentioned defects. Summary of the invention

[0005] The purpose of the present invention is to provide a CHO cell and suspension process method that can effectively solve the above technical problems; the CHO cell and suspension process method have high subculture efficiency, good batch consistency, small footprint, low cost, and can be produced on a large scale.

[0006] The purpose of the present invention can be achieved by the following measures:

[0007] The first aspect of the present invention provides a target gene expression vector, which is constructed by adding the sequence shown in SEQ ID NO.1 to the 5' end of the nucleotide sequence encoding the target amino acid, adding a stop codon to the 3' end, and then connecting with a vector plasmid.

[0008] The target gene expression vector of the present invention has a HindIII restriction site added to the 5' end of the sequence shown in SEQ ID NO.1, and an EcoRI restriction site added to the 3' end of the stop codon, and then is double-digested with HindIII and EcoRI restriction endonucleases and ligated with a vector plasmid that has also been double-digested with HindIII and EcoRI via T4 ligase to form the target gene expression vector.

[0009] The restriction endonuclease or ligase described in the present invention may also be replaced by other enzymes commonly used in the art.

[0010] The vector plasmid described in the present invention is pEE12.4, which can be purchased from the market, for example, from Protin Biotechnology (Beijing) Co., Ltd., with the product number BiovectorpEE12.4.

[0011] In some embodiments, the target amino acid of the present invention is shown as SEQ ID NO. 4. The nucleotide sequence encoding the target amino acid can be obtained according to conventional methods in the art, for example, in a specific example, it is shown as SEQ ID NO. 3.

[0012] The second aspect of the present invention provides a modified CHO cell, which is formed by transfecting the target gene expression vector of the present invention into a host cell.

[0013] The transfection method described in the present invention is a conventional method in the art, for example, after linearization, it is transfected into a host cell, the transfected plasmid is a plasmid that has been linearized and identified, and the product after enzyme digestion is extracted by ethanol precipitation and used for plasmid transfection.

[0014] The host cell described in the present invention is a Chinese hamster ovary cell commonly used on the market, such as Chinese hamster ovary cell CHO-K1Q, which can be purchased on the market, for example, purchased from Zhongshan Kangtian Shenghe Biotechnology Co., Ltd., item number A13101.

[0015] The improved CHO cells of the present invention can be cryopreserved for use. When in use, take working cells, quickly immerse the cryopreservation tube in a water bath and shake it to melt it immediately, and then perform conventional operations, for example, the cell suspension can be sucked out into a 15ml centrifuge tube (10ml of preheated culture medium is added in advance), centrifuged at 1000 rpm for 5 minutes; the supernatant is sucked away, and 5ml of culture medium is added to the cells to resuspend them to form a modified CHO cell suspension.

[0016] The third aspect of the present invention provides a method for producing a target protein, comprising the following steps:

[0017] (1) The modified CHO cell suspension was inoculated into a shake flask for recovery culture, and the recovered cell suspension was collected and inoculated into a WAVE bioreactor for culture;

[0018] (2) The culture temperature of the WAVE bioreactor is adjusted, and batch feeding culture is performed. When the culture reaches the 14th day or the cell viability in the bioreactor is lower than 70%, the culture supernatant is harvested to obtain the target protein.

[0019] In some embodiments, the cell density to be revived in the shake flask in step (1) of the present invention reaches 1.5-2.5×10 6 cells / ml, and when the cell viability was >95%, the revived cell suspension was collected and inoculated into the WAVE bioreactor for culture.

[0020] In some embodiments, the shake flask recovery in step (1) of the present invention is performed by gradually scaling up the culture using cell factory culture bottles of 125 mL shake flasks, 250 mL shake flasks, 500 mL shake flasks, and 1 L shake flasks, respectively.

[0021] In some embodiments, the medium formula used for shake flask recovery in step (1) of the present invention is 6.7 g / L glucose, 2.5 x 10 -5 M MSX (Methionine Sulfoximine), 1.9 g / L sodium bicarbonate, 4 mM glutamine.

[0022] In some embodiments, the culture medium used in the WAVE bioreactor in step (1) of the present invention is a serum-free culture medium ActiPro and 2.5x10 -5 MMSX.

[0023] In some embodiments, the inoculation density of the WAVE bioreactor in step (1) of the present invention is 0.1 to 1×10 6 cells / mL.

[0024] In some embodiments, the culture parameters of the WAVE bioreactor in step (1) of the present invention are: temperature 36-38°C, rocking speed 9-11 times / min, pH 7.10-7.40, and carbon dioxide concentration 4.5-5.5%.

[0025] In some embodiments, adjusting the culture temperature of the WAVE bioreactor in step (2) of the present invention is specifically to reduce the culture temperature to 33° C. on the 6th day of culture.

[0026] In some embodiments, the batch feeding culture in step (2) of the present invention specifically includes adding Cell Boost 7a at 4% of the culture volume and Cell Boost 7b at 0.4% of the culture volume on the 3rd, 5th, 7th, 9th, 11th and 13th days of culture, and supplementing the glucose content to 6-8 g / L when the glucose content is lower than 3 g / L.

[0027] In some embodiments, the culture supernatant is harvested in step (2) of the present invention by tangential flow filtration.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The present invention improves the target gene expression vector to obtain a stable high-expression cell line and achieve a protein expression level of more than 1 g / L.

[0030] (2) The improved CHO cells described in the present invention can be applied to the WAVE bioreactor, which adopts non-invasive wave-like shaking mixing to avoid damage to cells by the stirring blade end and bubbling, and provides a mild, low-shear, high-dissolved-oxygen cell culture microenvironment, which is beneficial to improve cell status, increase cell density and yield. The culture bag has reserved multiple interfaces for ventilation, inoculation, harvesting, feeding, sterile sampling and perfusion culture, etc., so that sterile sampling can be performed at any time in a non-clean environment to monitor the cell culture status.

[0031] (3) The improved CHO cells described in the present invention can be applied to serum-free culture, effectively preventing bacterial contamination, reducing the interference of exogenous factors, and reducing the level of endotoxin and other related impurities in the cell expression products.

[0032] (4) The present invention adopts a method combining shake flask culture and WAVE wave bioreactor for cell culture, which simplifies the operation steps, reduces the occupation of culture space, and reduces the culture time, thereby improving the culture efficiency, effectively preventing bacterial contamination, and reducing the endotoxin level in the product.

[0033] (5) The present invention uses tangential flow filtration to harvest the supernatant. Compared with dead-end filtration (where the flow direction is consistent with the filtration direction), the tangential flow filtration membrane has less particle accumulation on its surface and a more stable filtration speed.

[0034] (6) The improved CHO cells and their culture process described in the present invention greatly shorten the production cycle of the original adherent culture process from 65 to 80 days to 21 to 30 days (including cell recovery and expansion time), thereby reducing the use of manpower, material resources, and energy consumption, and reducing the risks of pollution, confusion, equipment failure, etc. caused by the long production cycle.

[0035] (7) The improved CHO cells and the culture process thereof described in the present invention are simple to culture, have low long-term costs, are easy to observe, do not require trypsin digestion, are easy to scale up for commercial production, have fewer operating steps in the production process, are highly automated, have high efficiency in subculture, and have good batch-to-batch consistency. The growth density is not limited by attachments. The production batches can be increased without increasing the plant area, effectively improving the plant utilization rate and increasing the production capacity, and the minimum supernatant production scale can reach 1000 L. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 It is the result of enzyme digestion; Figure 2 is the growth curve of viable cell density in WAVE culture;

[0037] Figure 3 is the cell viability curve of WAVE culture;

[0038] Figure 4 is the protein expression level. DETAILED DESCRIPTION

[0039] The following examples are provided for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0040] Example 1 Construction of improved CHO cells

[0041] (1) Construction of target gene expression vector: Codon optimization was performed according to the target amino acid sequence of tPA, and a HindIII restriction site and the sequence shown in SEQ ID NO.1 were added to the 5' end of the optimized sequence, and a stop codon and an EcoRI restriction site were added to the 3' end. Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to perform gene synthesis to form a sequence shown in SEQ ID NO.2, which was then double-digested with HindIII and EcoRI restriction endonucleases and ligated with a vector plasmid (Prudin Biotechnology (Beijing) Co., Ltd., product number BiovectorpEE12.4) that was also double-digested with HindIII and EcoRI restriction endonucleases via T4 ligase to form a target gene expression vector.

[0042] (2) Plasmid linearization and identification: After obtaining the recombinant expression vector plasmid, use PvuI enzyme digestion for linearization identification. The digestion product is extracted by ethanol precipitation and used for plasmid transfection. The digestion results are as follows: Figure 1 As shown, the results show that the target fragment after single enzyme digestion of the plasmid is single and consistent with the theoretical size. The concentration of the linearized plasmid is 1456 ng / L, and A260 / A280=1.89.

[0043] (3) Stable transfection of expression plasmid into CHO-K1Q cells:

[0044] The plasmid carrying the target gene was transfected into the host cell CHO-K1Q by electroporation. The host cell CHO-K1Q was revived with CD04 medium containing 4 mM glutamine, and 1×10 7 Host cell suspension (viable cell density 2.09×10 6cells / mL, cell viability 98.07%), centrifuged and discarded the supernatant, and resuspended with 779uL EX-CELL CD CHO Fusion. 30g of linearized plasmid and host cell suspension were fully mixed and added to the electroporation cup. The transfection conditions were: square wave voltage 300V, pulse time 15ms, 2 electric shocks, and 1s interval.

[0045] (4) Use MSX for pressurized screening, spread 96-well plate Minipool, expand the culture step by step according to the expression level of the well plate, and screen out cells with the best cell growth, expression and quality; use a single cell printer and monoclonal imaging system to sort and image the obtained Min-pool, expand step by step according to the expression level of the well plate, screen out monoclones with the best cell growth, expression and quality, establish an original cell library and test for sterility and mycoplasma. The monoclones are continuously pressurized to PDL30 and then depressurized to PDL60 after PDL30, and the growth and expression stability of cells at different time points of PDL0 and PDL60 are investigated. Improved CHO cells are formed.

[0046] Example 2

[0047] (1) Immerse the cryopreservation tube containing the modified CHO cells of Example 1 in a 37°C water bath and shake to thaw immediately; aspirate the cell suspension into a 15 ml centrifuge tube (pre-added with 10 ml of preheated CD04 medium), and centrifuge at 1000 rpm for 5 minutes; discard the supernatant, add 5 ml of CD04 medium containing 4 mM glutamine to the cells for resuspending; add 25 ml of CD04 medium containing 4 mM glutamine to a 125 ml shake flask, and culture at 5% CO2, 37°C, and 120 rpm.

[0048] (2) The cell density reaches 1.5-2.5×10 6 cells / ml, dilute the above cell suspension to 3-5×10 5 cells / ml was added to a 250 mL shake flask, and 60 ml of CD04 medium (components: 6.7 g / L glucose, 2.5 x 10 -5 M MSX, 1.9 g / L sodium bicarbonate, 4 mM glutamine), and cultured in a 250 mL shake flask in a carbon dioxide shaking incubator at 5% CO2, 37°C, and 120 rpm.

[0049] (3) When the cell density reaches 1.5-2.5×10 6 When the cell suspension in the 250mL shake flask was 3-5×10 cells / ml, a 250mL shake flask was taken out from the carbon dioxide shaking incubator, wiped and disinfected, and then placed under the laminar flow hood. The cell suspension in the 250mL shake flask was diluted to 3-5×10 5cells / ml were added to a 500mL shake flask; at the same time, 100ml of expansion medium was added to the 500mL shake flask, the lid was closed, numbered, and the 500mL shake flask was placed in a carbon dioxide shaking incubator at 5% CO2, 37°C, and 120rpm for culture. The culture medium was CHO CD04 liquid culture medium (components were 6.7g / L glucose, 2.5x10 -5 M MSX, 1.9 g / L sodium bicarbonate, 4 mM glutamine).

[0050] (4) After 3-5 days, the cell density reaches 1.5-2.5×10 6 cells / ml, dilute the culture medium in the cell shake flask to 3-5×10 5 cells / ml was added to a 1L shake flask; at the same time, 200 ml of expansion medium was added to the 1L shake flask, the lid was covered, numbered, and the 1L shake flask was placed in a carbon dioxide shaking incubator with 5% CO2, 37°C, and 120 rpm for culture. The culture medium was CHO CD04 liquid culture medium (components were 6.7 g / L glucose, 2.5 x 10 -5 M MSX, 1.9 g / L sodium bicarbonate, 4 mM glutamine).

[0051] (5) After 3-5 days, the cell density reaches 1.5-2.5×10 6 cells / ml; under sterile conditions, dilute the culture medium in the cell shake flask to 3-5×10 5 cells / ml were added to 5 1L shake flasks; at the same time, 200ml of expansion medium was added to the 1L shake flasks, the flasks were capped, numbered, and placed in a carbon dioxide shaking incubator with 5% CO2, 37°C, and 120rpm. The culture medium was CHO CD04 liquid culture medium (components were 6.7g / L glucose, 2.5x10 -5 M MSX, 1.9 g / L sodium bicarbonate, 4 mM glutamine).

[0052] (6) After 3-5 days, when the cell density reaches 1.5-2.5×10 6 cells / ml, and the cell viability was >95%, then all the cell suspension in the cell shake flask was transferred to the 50LWAVE wave bioreactor (working volume 25L) of Guangzhou Abetech Biotechnology Co., Ltd. for culture under sterile conditions, with an inoculation volume of 1.0-1.5×10 10 cells, set the culture conditions as follows: temperature 37±1℃, rocking speed 10 times / min, pH 7.10-7.40, carbon dioxide concentration 5%, serum-free culture medium ActiPro, and add 2.5x10 -5M MSX, for use with Cell Boost 7a and Cell Boost 7b feed media.

[0053] (7) Adjusting the culture temperature of the WAVE bioreactor and performing batch feeding culture, as shown in Table 1, where the addition amounts of feed 1 and feed 2 are calculated based on the volume percentage of the culture volume:

[0054] Table 1

[0055]

[0056] (8) When the cell viability in the bioreactor is lower than 70%, the culture supernatant is harvested, and the harvested supernatant is filtered by tangential flow to obtain a protein supernatant, which is purified to obtain a protein harvest liquid. The protein is consistent with the standard after comparison, and the sequence is shown in SEQ ID NO. 4. The tank culture cycle does not exceed 21 days, and the total harvest volume is determined according to the cell growth situation, and the usage amount is more than 1000L.

[0057] The growth curve of viable cell density in this example is as follows Figure 2 As shown, the highest value is 1.6×10 7 cells / mL. The cell viability was still above 80% at the time of harvest. Figure 3 When harvested, the cell expression level was at 1 g / L, far exceeding the 30 mg / L level of the adherent process. Figure 4 shown.

Claims

1. A target gene expression vector, characterized in that: The target gene expression vector is constructed by adding the sequence shown in SEQ ID NO.1 to the 5' end of the nucleotide sequence encoding the target gene, adding a stop codon to the 3' end, and then connecting with the vector plasmid.

2. The target gene expression vector according to claim 1, characterized in that: A HindIII restriction site was added to the 5' end of the sequence shown in SEQ ID NO.1, and an EcoRI restriction site was added to the 3' end of the stop codon. Then, after double digestion with HindIII and EcoRI restriction endonucleases, the vector was ligated with a vector plasmid that was also double digested with HindIII and EcoRI via T4 ligase to form a target gene expression vector.

3. The target gene expression vector according to claim 1, characterized in that: The vector plasmid is pEE12.

4.

4. The target gene expression vector according to claim 1, characterized in that: The target amino acid is shown as SEQ ID NO.4, and the nucleotide sequence encoding the target amino acid is shown as SEQ ID NO.

3.

5. A modified CHO cell, characterized in that: The target gene expression vector according to any one of claims 1 to 4 is transfected into a host cell.

6. The improved CHO cell according to claim 5, characterized in that The host cell is Chinese hamster ovary cell CHO-K1Q.

7. A method for producing a target protein, characterized in that: The following steps are involved: (1) The modified CHO cell suspension was inoculated into a shake flask for recovery culture, and the recovered cell suspension was collected and inoculated into a WAVE bioreactor for culture; (2) The culture temperature of the WAVE bioreactor is adjusted, and batch feeding culture is performed. When the culture reaches the 14th day or the cell viability in the bioreactor is lower than 70%, the culture supernatant is harvested to obtain the target protein.

8. The method for producing the target according to claim 7, characterized in that: The cell density in the shake flask to be revived in step (1) reaches 1.5-2.5×10 6 cells / ml, and when the cell viability is greater than 95%, the recovered cell suspension is collected and inoculated into a WAVE bioreactor for culture; preferably, in step (1), the shake flask recovery is performed by using 125mL shake flasks, 250mL shake flasks, 500mL shake flasks, and 1L shake flasks for cell factory culture flasks for step-by-step amplification culture; more preferably, the culture medium formula used for shake flask recovery in step (1) is 6.7g / L glucose, 2.5x10 -5 M MSX, 1.9 g / L sodium bicarbonate, 4 mM glutamine.

9. The method for producing a target protein according to claim 7, characterized in that: The culture medium used in the WAVE bioreactor in step (1) is serum-free culture medium ActiPro and 2.5x10 -5 M MSX; Preferably, the inoculation density of the WAVE bioreactor in step (1) is 0.1 to 1×10 6 cells / mL; more preferably, the culture parameters of the WAVE wave bioreactor in step (1) are: temperature 36-38°C, rocking speed 9-11 times / min, pH 7.10-7.40, and carbon dioxide concentration 4.5-5.5%.

10. The method for producing a target protein according to claim 7, characterized in that: In step (2), adjusting the culture temperature of the WAVE bioreactor is specifically to reduce the culture temperature to 33°C on the 6th day of culture; preferably, in step (2), the batch feeding culture is specifically to add CellBoost 7a at 4% of the culture volume and CellBoost 7b at 0.4% of the culture volume on the 3rd, 5th, 7th, 9th, 11th and 13th days of culture, and in addition, when the glucose content is lower than 3g / L, it is supplemented to 6-8g / L; more preferably, in step (2), the culture supernatant is harvested by tangential flow filtration.

Images