WO2018032464A1 - High-throughput cell line screening method for cho-dhfr expression system - Google Patents

Abstract

A high-throughput cell line screening method for a CHO-Dhfr expression system. The method comprises: performing three rounds of methotrexate stress treatment, selecting a cell pool with 40-50% cell viability, and performing monoclonal conversion in a specific semi-solid nutrient medium.

Description

High-throughput screening method for cell line of CHO-Dhfr expression system Technical field

The present invention relates to the field of cell engineering, and in particular to a high throughput screening method for a CHO-Dhfr expression system cell line.

technical background

"Engineering cells" for the production of monoclonal antibodies or recombinant protein drugs with a relatively large molecular weight are required to have the following characteristics: good growth characteristics, high serum-free suspension culture density (1 to 2 × 10 ⁷ cells/ml); heterologous protein Strong expression ability (20～70pg/cell/day, pcd), with correct post-translational modification ability; host and recombinant cell lines have clear genetic background, stable phenotype, and meet relevant regulatory requirements.

Currently, one of the most commonly used screening systems in the construction of cell lines in the industry is the CHO-Dhfr system. CHO cells are Chinese Hamster Ovary, DHFR refers to dihydrofolate reductase, which catalyzes the conversion of dihydrofolate to tetrahydrofolate, DHFR knockout CHO cells (eg Invitrogen DG44) It is not possible to synthesize tetrahydrofolate and can only grow in medium supplemented with thymidine, glycine and guanidine. The system inserts a foreign gene downstream of the expression vector containing the DHFR gene and transfers it to the host cell DG44 cells, so that it can be grown in a medium free of thymidine, glycine and sputum. In the cell culture, DHFR inhibitor methotrexate (MTX) is added to amplify the MTX gene and the target gene linked thereto, thereby increasing the copy number of the target gene, thereby increasing the expression level of the target gene.

The purpose of cell line screening is to obtain stable and high-yielding cell lines, and the measurement criteria include antibody expression amount, product quality, metabolic stability, cell matrix stability and the like. Conventional screening methods are: construction of expression constructs → transfection into host cells → cell selection by pressure screening → cell pooling of cell pools → expansion of high-expression cell lines and screening → stability of cell lines Evaluation.

The cell pool is routinely obtained by using a multi-round pressurization strategy when the expression construct is transfected into the host cell until the increase in expression is not significant, and the previous round is the highest compression cycle. After resuscitating the cells, the cell pool is determined after the cell viability is >95%, or the cells with the expression level of TOP2 to 5% are flow-selected by the cell pool mentioned above, and the cell viability is again >95%. It is the cell pool. In general, in the same culture environment, low-expression cell lines use nutrients more for cell growth, so they grow faster, while high-expression cell lines grow slower. Therefore, the above two vigors are > In the process of gradually recovering cell viability in 95% of cell pools, most of the high-expression cell lines are slower and slower to lose; while the low-expressing cell lines are longer and faster; eventually, the cell pool is highly expressed. The low expression of the cell line is high. At the same time, in the process of cell viability recovery, increase the number of cell passages. According to experimental experience, each time the cell viability is restored, the cells increase by 15-20 Generations (Generation, generations, cells are multiplied once, called 1Generation), affecting cell lines. stability.

The most common method for monoclonal screening is limiting dilution cloning (LDC), which is low-cost and easy to operate. The cell density is placed in a 96-well plate at <1cell/well. The medium is a liquid medium, most of the time. Adding a certain proportion of conditioned medium (containing cell growth factor) as required, the cell growth state is extremely high, the cell pool with <90% viability is almost difficult to survive, and the cloning efficiency is extremely low, even if the monoclonalization activity is >95%. Cell pool, the highest cloning efficiency is only about 7%, the average level is 3% to 4%, and the operation is cumbersome, the workload is large, the experimental period is long, the experimental efficiency is low, and high-throughput screening cannot be achieved.

Monoclonal screening was carried out by semi-solid medium method, in which the cell pool was spread on a semi-solid medium at a certain density to form a single clone, which was then selected to gradually expand the culture and screen the highly expressed cell line. At present, high-throughput screening instruments such as ClonePix2 are being used for high-throughput screening on various semi-fixed media. However, with reference to existing literature, how to improve the colony formation rate and to filter out stable high tables faster and easier. The cell line has always been a technical difficulty in the art.

Summary of the invention

The object of the present invention is to provide a high-throughput screening method for a CHO-Dhfr expression system cell line, which has a high colony formation rate and is easy to screen out stable and high expression cell lines. Specifically, the method overcomes the problem of extremely low cloning efficiency by limiting dilution method by selecting cell viability in a 40% to 50% cell pool and performing monoclonalization on a semi-solid medium while utilizing a high-throughput cell screening system. Therefore, it is easier to screen out high-quality cell lines with stable high expression.

A high-throughput screening method for a CHO-Dhfr expression system cell line, comprising the steps of: (1) constructing an expression construct, (2) transfecting into a host cell, and (3) obtaining a cell pool by MTX pressurization screening, 4) The cell pool is monoclonalized on a semi-solid medium, (5) the cell strain is screened using a high-throughput cell screening system, and (6) the stability of the cell strain is assessed. It is characterized in that the cell viability of the cell pool described in the step (4) is 40% to 50%.

According to a specific embodiment of the present invention, the MTX pressure screening according to the step (3) is divided into three rounds, and the concentration of the MTX is 100 nM, 1000 nM, 5000 nM in sequence;

According to some embodiments of the present invention, the step (4) is performed on a semi-solid medium, wherein the final concentration of the cells in the semi-solid medium is 150 cells/mL;

According to some embodiments of the invention, the semi-solid medium of step (4) contains a conditioned medium and methylcellulose. Preferably, the conditioned medium comprises 10% of the total volume of the semi-solid medium. Preferably, the content of methylcellulose in the semi-solid medium is from 0.9 to 1.5 g/L. According to some embodiments of the present invention, the conditioned medium is prepared by taking a stable cell strain that has been transfected with pOptiVEC (the vector containing the Dhfr gene, which does not contain other foreign proteins) with the addition of 8 mM glutamine. Batch culture was carried out in OptiCHO Medium, and the cells were cultured to a density of 0.9×10 ⁷ to 1.1×10 ⁷ cells/mL and the cell viability was >95%. The supernatant and cells were separated by centrifugation, and the supernatant was filtered. Wherein the cell strain is DG44 cells.

According to some embodiments of the present invention, the semi-solid medium according to the step (4) further comprises a 1% by volume fluorescent antibody selected from the group consisting of CloneDetect-anti-human IgG (H+L) detection.

According to some embodiments of the invention, the high throughput cell screening system of step (5) is ClonePix2 of Molecular Devices.

According to some embodiments of the invention, the Dhfr expression system is transfected into an expression vector carrying a Dhfr gene and inserted with a foreign gene downstream of the Dhfr gene into a CHO cell (DG44) that knocks out the Dhfr gene.

Explanation of terms

Dihydrofolate reductase (DHFR) refers to the reduction of dihydrofolate by reducing coenzyme II (NADPH) to produce tetrahydrofolate oxidoreductase. Tetrahydrofolate is involved in the synthesis of purines and pyrimidines, which promotes the formation of normal blood cells. The inhibitor of DHFR is methotrexate (MTX).

Methotrexate (MTX) pressurization is a gene for the DHFR expression system to amplify the downstream gene of the Dhfr gene, thereby realizing the foreign gene. High expression, in the present invention, three rounds of large pressure, namely: 100 nM, 1000 nM, 5000 nM, to obtain a cell pool with a lower viability under 5000 nM MTX pressurization.

After the neomycin resistance gene (Neo) is integrated into the DNA of eukaryotic cells, it can initiate the transcription of the sequence encoded by the Neo gene into mRNA, thereby obtaining high-efficiency expression of the resistance product aminoglycoside phosphotransferase and making the cells resistant. It can be grown in a selective medium containing G418 containing a neomycin resistance gene.

The semi-solid medium used in the present invention is a semi-solid medium containing 10% conditioned medium. Conditioned media provide nutrients for cell growth that help clone formation. The conditioned medium added to the present invention is self-made in the laboratory, and the preparation method is described in the examples. The semi-solid medium used in the present invention is a semi-solid medium containing methyl cellulose. The addition of methylcellulose enables the same monoclonal to form a cell cluster upon cleavage, and enables the antibodies secreted by the cells to accumulate around the cell cluster with fluorescent antibodies (CloneDetect-anti-human IgG (H+L) detection (Molecular Devices) , item number K8200)) combined to achieve the purpose of quantitative testing. In addition to the commercially available medium CloneMatrix (Molecular Devices, Cat. No. K8510), methylcellulose solution can also be prepared using methylcellulose powder (CAS#: 9004-67-5). When the plate is mixed, the methyl cellulose solution and the basic medium are mixed in a certain ratio to be the semi-solid medium used in the present invention, and finally, the content of methyl cellulose in the semi-solid medium is 0.9 to 1.5 g/mL. The basal medium is selected from the group consisting of OptiCHO Medium (Invitrogen, 12681-011).

The high-throughput cell screening system used in the present invention is ClonePix2 of Molecular Devices, and the screening process is that ClonePix2 images the cells→ClonePix2 calculates the fluorescence intensity of the clone→ClonePix2 picks up the monoclonal clone with high fluorescence intensity→96-well plate to continue the culture. The monoclonal antibody with high fluorescence intensity represents a monoclonal cell strain with high expression level, because the labeled fluorescent antibody acts as a secondary antibody, and binds to the antibody secreted by the cell strain, and the fluorescent cell antibody which binds with a high expression amount has many fluorescent antibodies. The intensity is high.

The stability of the cell line was evaluated by continuously screening the highly expressed cell lines to the 60th generation, and the cells of the 20th, 40th and 60th generations of each cell line were evaluated for 500mL batch culture. Comparing the expression levels of the cell lines in different generations, according to the percentage decrease of the expression of the cell line, the stable level of the cell line was determined: within 10%, it was stable; within 30%, stable; more than 30%, unstable.

The technical benefits of the method of the invention are as follows:

(1) High-throughput screening of CHO-Dhfr expression system cell lines: This screening method uses the ClonePix2 high-throughput screening system, which is 50 times more efficient than the screening with conventional limiting dilution monoclonalization. The screening cycle was shortened by at least 1/3, mechanized, and more than 95% of low-yielding clones were directly excluded.

(2) High colony formation rate and improved screening success rate: the cell pool with >95% cell viability with conventional limiting dilution, the cloning efficiency is usually 3-4%, and the cell viability is 40% using this screening method. In the 50% cell pool, the cloning efficiency of adding and conditioned medium was about 60% and 100%, respectively, and the improvement in cloning efficiency was large.

(3) High expression level: It is easier to screen out more highly expressed cell lines than screening using a cell pool with a cell viability of >95%.

(4) Good stability: cell lines obtained by monoclonalization of cell cells with cell viability in 40% to 50%, compared with cell lines screened by cell pools with >95% viability, stability Significantly increased.

DRAWINGS

Figure 1 shows the antibody expression of each group of clones in the 96-well plate culture stage during the monoclonal screening process of cell pool A (viability 41.58%), cell pool B (vigorization 49.66%) and cell pool C (viability 95.86%). a comparison map of the distribution;

Figure 2 shows the antibody expression of each group of clones in the 6-well plate batch culture stage during the monoclonal screening process of cell pool A (viability 41.58%), cell pool B (vigorization 49.66%) and cell pool C (viability 95.86%). Comparison map of quantity distribution;

Figure 3 is the expression level of each group of clones in the 500mL shake flask batch culture stage during the monoclonal screening process of cell pool A (viability of 41.58%), cell pool B (vigorization of 49.66%) and cell pool C (viability of 95.86%). A comparison chart of the distribution.

detailed description

The invention is further described in detail below with reference to the preferred embodiments thereof, which are set forth to illustrate the invention and not to limit the scope of application of the invention. The reagents, culture media, consumables, and the like used in the experiment can be obtained by a commercially available route.

Example 1 Preparation of a cell pool

1. Construction of expression constructs

Refer to OptiCHO ^TM Antibody Express Kit (purchased from Invitrogen), pOptiVEC ^TM -

TA Cloning Kit (purchased from Invitrogen) and pcDNA ^TM 3.3-

TA Cloning Kit (purchased from Invitrogen) instructions for the preparation of expression vectors for the expression of heavy and light chains of anti-TNFα full human antibody (expressed under the item number A- before the vector name), respectively: A- pOptiVEC-VL (containing Dhfr-VL) and A-pcDNA3.3-VH (containing Neo-VH).

2. Transfection into host cells

use

HiPure Plasmid DNA Purification Kit (purchased from Invitrogen) was used to extract A-pOptiVEC-VL and A-pcDNA3.3-VH of Anti-TNFα full human antibody, which were linearized and purified by PvuI (purchased from TaKaRa), respectively. The same amount of confluence, taking a total of 40μg DNA / electroporation system, using electroporation (Bio-Rad Gene PulserXcell Eukaryotic System, setting parameters: 300V, 900μF, pulse time 20ms) electric shock transfection of DG44 cells in logarithmic growth phase (10 ⁷ cells) (Invitrogen, OptiCHO ^TM Antibody Express Kit), resuspended the cells after three shocks in 12 mL of 8 mM L-glutamine OptiCHO Medium, and added to a 6-well culture plate at 2 mL/well. The culture was allowed to stand at 37 ° C under 5.0% CO ₂ to restore the mechanical damage of the cells caused by the electric shock. The medium was mixed with L-glutamine (purchased from Invitrogen) mother liquor and OptiCHO Medium (purchased from Invitrogen). If 12 mL of medium was used, 0.5 mL of 200 mM L-glutamine mother liquor was added to 11.5 mL. In the OptiCHO Medium, the preparation method of the following 8 mM L-glutamine OptiCHO Medium is the same as here.

After the cells were cultured for 48 hours, the cells were centrifuged, and the cells were resuspended in 30 mL of OptiCHO Medium containing 500 μg/mL G418 and 8 mM L-glutamine, and shake cultured at 140 rpm, 37 ° C, 8.0% CO ₂ for three days. The first generation (Passage), cultured for 3 generations, the cell viability recovered to over 80%. At this time, the DG44 cell pool Y with stable transfection double vector (A-pOptiVEC-VL, A-pcDNA3.3-VH) was obtained (viability was 83.21). %). G418 in the medium was from 50 mg/mL G418 mother liquor (purchased from Invitrogen), and was added to OptiCHO Medium in proportion when used. Due to the small addition volume, only 1% of the final volume, the final volume increased after addition. Ignorable.

3. MTX pressure screening to obtain cell pool

The DG44 cell pool Y stably transfected with A-pOptiVEC-VL and A-pcDNA3.3-VH was subjected to three-stage pressurization with MTX, and cultured under shaking at 37 ° C and 8.0% CO ₂ . The concentration of the pressurization was: 100 nM, 1000nM, 5000nM. During the MTX pressurization process, each concentration was subjected to centrifugation for 3 to 5 passages. When the cell viability was restored to 70% or more and the cell density reached 3×10 ⁵ /mL, the MTX concentration was increased to the next gradient. After subculture, the cell supernatant was taken, and the expression of anti-TNFα whole human antibody was determined by Fortebio (PALL, QKe). The concentration of MTX (purchased from Sigma) was diluted from 1 mM MTX mother liquor. The preparation method of 1 mM MTX mother liquor was as follows: 10 mg of MTX powder was dissolved in 22 mL of PBS solution, and 0.22 μm filter was filtered and sterilized, 0.5 mL/ The tube is divided and stored at -20 ° C for use. It is thawed at room temperature during use and can be stored at 4 ° C for one month after thawing. When used, it is added to the medium in proportion. Since the addition volume is small, it is only 0.01 to 0.5% of the final volume. After the addition, the increase of the final volume is negligible.

When the pressure is raised to the last concentration of 5000 nM, the cell pool with cell viability of 40% to 55% is selected, and cell pool A (viability is 41.58%), cell pool B (viability is 52.66%), and cell pools A and B are obtained. The amount of antibody expressed per unit cell time has increased by 125 to 135 times compared to cell pool Y. Continue to maintain the pressure of 5000 nM until the vitality recovered to more than 90%. Take the cell bank C (the viability is 95.86%) at this time as the experimental control group.

Example 2 Effect of conditioned medium on colony formation

1. Preparation of conditioned medium and semi-solid medium

Preparation of conditioned medium: A stable cell line transfected with pOptiVEC (the vector containing the Dhfr gene, which does not contain other foreign proteins) was batch cultured in 300 mL of 8 mM glutamine OptiCHO Medium, and the cells were cultured to density. When the cell viability was between 0.9×10 ⁷ and 1.1×10 ⁷ cells/mL and the cell viability was >95%, the supernatant and cells were separated by centrifugation, and the supernatant was filtered through a 0.22 μm membrane to obtain 10 mL/tube, 25 mL/ Tube, 50 mL / tube was dispensed, and stored at -80 ° C until use. Estimated dosage at the time of use Select the appropriate volume of conditioned medium at 37 ° C, thaw in a water bath, and discard the rest.

Preparation of semi-solid medium: The ingredients in the semi-solid medium formula of Table 1 were vigorously mixed at room temperature according to the dosage of the preparation system 1, 2, and wait for the bubbles to disappear slowly.

Among them, Clonematrix is a semi-solid medium matrix purchased from Molecular Devices;

CloneDetect-anti-human IgG (H+L) detection is a fluorescent antibody purchased from Molecular Devices;

Preparation of 1 mM MTX mother liquor: 10 mg of MTX powder was dissolved in 22 mL of PBS solution, and 0.22 μm filter was filtered and sterilized;

2.5×OptiCHO Medium preparation: Weigh 4.825g CD OptiCHO ^TM AGT ^TM Medium powder (purchased from Invitrogen) to 85mL ultrapure water, stir at room temperature for 30min, then dilute to 100mL with ultrapure water to adjust pH 7.4～7.8 .

Table 1 Semi-solid medium formula for monoclonal cell pool

Semi-solid medium formula	Formulation system 1	Formulation system 2
Clonematrix	12mL	12mL
CloneDetect-anti-humanIgG(H+L)detection	0.3mL	0.3mL
Sterile water	4.35mL	1.35mL
Conditioned medium	--	3mL
1mMMTX mother liquor	0.15mL	0.15mL
200 mM aqueous glutamine solution	1.2mL	1.2mL
2.5×OptiCHOMedium	10.5mL	10.5mL

2. Spread cell pool A to semi-solid medium

A suitable amount of cells from cell pool A (viability 41.58%) were centrifuged and resuspended in 4.0 mL of 2.5×OptiCHO Medium (see the preparation procedure of step 1) to obtain a cell suspension having a cell density of 3000 cells/mL.

1.5 mL of the cell suspension of the above cell pool A was mixed with the semi-solid medium of the preparation system 1, and gently mixed, and then added to 2 6-well culture plates at 2 mL/well, and named as AK(1) to (2) ), static culture for 10 to 12 days, 37 ° C, 5.0% CO ₂ conditions, until the cell mass is slowly formed; after the remaining cell suspension is thoroughly mixed, take 1.5 mL and the semi-solid culture of the preparation system 2 The base was mixed and gently mixed, and added to 2 6-well culture plates at 2 mL/well, and named as AK(3)-(4), static culture for 10 to 12 days, 37 ° C, 5.0% CO ₂ . Wait until the cell mass slowly forms.

3.Clonepix imaging

After the cells were cultured for 12 days in 6-well plates AK(1) to (4), cell clusters with uniform distribution and moderate size (0.3-0.6 mm ² ) were visible to the naked eye. At this time, ClonePix2 System (Molecular Devices) could be used to select high fluorescence intensity. Cloning.

(1) According to the ClonePix2 operating instructions, the instrument is gradually Prepare For Pick Run, this process to ensure that the needle is correctly fired, the camera, needle and microplate are matched, the liquid system is sterile and ready for use.

(2) Then run Pick Run, the process is: including settings (including imaging settings, pick settings, sterilization settings, clone identification settings).

(3) AK (1) to (4) were imaged respectively, and the cloning formation efficiency is shown in Table 2.

Table 2 compares the formation of conditioned medium clones

Comparison project	Formulation system 1	Formulation system 2
6-well plate number	AK(1)~(2)	AK(3)～(4)
Total number of cells	3600	3600
Number of imaging clones	2299	3569
Clonal formation rate	63.9%	99.1%

It can be seen from Table 2 that the cell pool A is placed on the semi-solid medium, and the formation rate of the experimental group added with 10% conditioned medium is close to 100%, and the conditioned medium is not configured. 1 The experimental group clone formation rate was only about 60%.

Example 3 Monoclonal screening for high expression stable cell lines

1. Cell pool A, B, C monoclonalization

Prepare 3 parts of semi-solid medium according to the preparation system 2 of Table 1, and take appropriate cells of cell pool A (viability 41.58%), cell pool B (vigorization 49.66%) and cell pool C (viability 95.86%), and resuspend separately. Three cell suspensions with a cell density of 3000 cells/mL were obtained in 1.5 mL of 2.5 x OptiCHO Medium. Three cell suspensions were separately mixed with three semi-solid media, gently mixed, and added to two 6-well culture plates at 2 mL/well, designated as AK(5)-(10), and static culture 10 ~12 days, 37 ° C, 5.0% CO ₂ conditions, the cell mass slowly formed.

2.ClonePix2 imaging and picking clones

After the cells were cultured for 12 days in 6-well plates AK(5)-(10), cell clusters with uniform distribution and moderate size (0.3-0.6 mm ² ) were visible to the naked eye. Fluorescence intensity could be selected by ClonePix2 System (Molecular Devices). High clones.

(1) According to the ClonePix2 operating instructions, the instrument is gradually Prepare For Pick Run, this process to ensure that the needle is correctly fired, the camera, needle and microplate are matched, the liquid system is sterile and ready for use.

(2) Then run Pick Run, the process is: including settings (including imaging settings, pick settings, sterilization settings, clone identification settings).

(3) The imaging results of AK(5)-(10) were analyzed. The analysis was based on setting the thresholds of cell size, regularity and fluorescence intensity, and grouping the clones. Among them, the regularity of the cell mass is measured by the perimeter/area ratio and the smoothness of the cell edge. The purpose is to make the shape of the cell cluster close to a circle; the fluorescence degree of the cell cluster includes various algorithms, for example: external Average Mean Intensity, External Median Intensity, External Total Intensity, Internal Intensity SD, Internal Mean Centre Intensity, Internal Mean Intensity, Internal Mean Intensity Intensity, Internal Median Intensity, Internal Total Intensity, Normalized Intensity, and SumTotal Intensity. According to the secretory expression of CHO cells, the present invention uses external average fluorescence intensity as a standard. Cell lines were screened.

(4) Select a cell line with strong external average fluorescence intensity from AK(5)~(10), and select 300 strains from each cell pool to be picked to the target plate 96-well plate, and each cell-derived cell is 5 Block plates, named AKC96 (1) ~ (15), see Table 3.

(5) End the procedure and place the target plate cells AKC96(1)～(15) in the incubator for cultivation. Generally, the cloned confluence can be as long as 15% or more after 5-7 days.

Table 3: Comparison of monoclonal clone formation rate and external average fluorescence intensity in different cell pools

3. Expanded culture and screening of clones

The process of clonal expansion and screening was as follows: (1) 300 clones/cell pools were selected according to the external fluorescence intensity, and picked up to 5 96-well plates for culture. The expression distribution range is shown in Figure 1; (2) Screening In the step (1), the 48 clones/cell pools with relatively high expression amount were transferred to a 6-well plate for expansion, and the distribution range of the expression expression of the batch culture is shown in Fig. 2; (3) the expression amount in the step (2) was relatively selected. The higher 10 clones/cell pools were transferred to a 500 mL shake flask for expansion, and the distribution range of the batch culture expression is shown in Fig. 3. Among them, the histogram shows the number of clones in a certain range of antibody expression, and the curve shows the range and frequency of the distribution of clone expression; from the trend graph of each expansion stage, it can be seen that the cell pools A and B are highly expressed. The amount of strain is more.

4. Stability of cell lines

The cell lines derived from cell pools A, B, and C, respectively, were expanded to the shake flask size and named as: cell pool A: clones (1) to (10), cell pool B: clones (11) to ( 20), cell pool C: clones (21) to (30).

The cell lines of clones (1) to (30) were further passaged to the 60th generation, and the cells of the 20th, 40th and 60th generations of each cell strain were taken and evaluated for 500mL batch culture. Comparing the expression levels of the cell lines in different generations, according to the percentage decrease of the expression of the cell line, the stable level of the cell line was determined: within 10%, it was stable; within 30%, stable; more than 30%, unstable. The results are shown in Table 4.

Table 4: Stability Evaluation Tables for Clones (1) to (30) Screened by Cell Pools A, B, and C

It can be seen from the above results that the strains selected by cell pools A and B have higher stability.

Based on the above experimental results, it can be seen that the cell pool with cell viability in 40% to 50% is selected for monoclonalization in a specific semi-solid medium, and the colony formation rate is significantly improved, and can be screened out in a short time. More strains with high expression and stability.

Claims (10)

1. A high-throughput screening method for a CHO-Dhfr expression system cell line, comprising the steps of: (1) constructing an expression construct, (2) transfecting into a host cell, and (3) obtaining a cell pool by pressure-removing a methotrexate, ( 4) the cell pool is monoclonalized on a semi-solid medium, (5) the cell strain is screened by a high-throughput cell screening system, and (6) the stability evaluation of the cell strain; characterized by the step (4) The cell viability of the cell pool is 40% to 50%.
2. The method according to claim 1, wherein the methotrexate press screening according to the step (3) is divided into three rounds, and the concentration of methotrexate is 100 nM, 1000 nM, and 5000 nM, respectively.
3. The method according to claim 1, wherein the step (4) is carried out on a semi-solid medium, wherein the final concentration of the cells in the semi-solid medium is 150 cells/mL.
4. The method according to claim 1, wherein the semi-solid medium according to the step (4) contains a conditioned medium and methyl cellulose.
5. The method of claim 4 wherein said conditioned medium comprises 10% of the total volume of the semi-solid medium.
6. The method according to claim 4, wherein the methylcellulose is present in the semisolid medium in an amount of from 0.9 to 1.5 g/L.
7. The method according to any one of claims 4 to 6, wherein the conditioned medium is prepared by batch-culturing a stable cell strain which has been transfected with the carrier in OptiCHO Medium supplemented with 8 mM glutamine, the cells When the density is between 0.9×10 ⁷ and 1.1×10 ⁷ cells/mL and the cell viability is >95%, the supernatant and the cells are separated by centrifugation, and the supernatant is filtered, and the vector contains the Dhfr gene, and does not contain Genes for other foreign proteins.
8. The method according to claim 7, wherein the vector is pOptiVEC and the cell line is DG44 cells.
9. The method according to claim 4, wherein the semi-solid medium according to the step (4) further contains a fluorescent antibody at a volume ratio of 1% by volume.
10. The method according to claim 1, wherein the expression construct of the step (1) is a vector for expressing an anti-TNFα full human antibody heavy and light chain.

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