KR101271713B1 - A transformed cell line transfected by a vector which over-expresses Bcl2 protein and a vector which produces shRNA against LDH-A, and a method of producing target proteins using the above transformed cell line - Google Patents

Abstract

The present invention is transformed to overexpress B-cell lymphoma 2 (Bcl2), an anti-destructive gene, and to low expression of a lactate dehydrogenase A (LDH-A) gene that catalyzes the production of lactic acid. A novel CHO cell line which lacks a dihydrofolate reductase (Dhfr) gene, a method for preparing the same, and a method for producing a target protein using the transformed CHO cell line, wherein the transformed CHO cell line Due to the overexpression of anti-satellite protein Bcl2, preliminary death is inhibited and cell viability is increased, and low expression of lactic acid-producing enzyme LDH-A inhibits the production of lactic acid, one of the waste products accumulated in the culture medium. Reduces the accumulation of lactic acid and converts two traits simultaneously by manipulating genes involved in cell survival and cell metabolism The conversion can be useful for the production of the desired target protein because it increases the cell survival rate and the flow of cell metabolism not only increases the productivity of the target protein but also improves the quality of the target protein produced by reducing stress on the external environment. Can be.

Description

A novel CHO cell line lacking a DHFR that overexpresses a Bc2 protein and expresses a short hairpin RNA against LHDH-A, a method for producing the same, and a method for producing a target protein using the transformed CHO host cell a vector which over-expresses Bcl2 protein and a vector which produces shRNA against LDH-A, and a method of producing target proteins using the above transformed cell line}

The present invention relates to a vector that overexpresses the anti-programmed cell death protein Bcl2 and a short hairpin RNA that inhibits the expression of LDH-A involved in catalyzing the production of lactate from pyruvate. The present invention relates to a novel transformed CHO cell line into which a vector expressing hairpin RNA) is introduced, a method for producing the same, and a method for producing a target protein using the transformed cell line.

In the field of biology or medicine, the desired protein can be obtained mainly by culturing the transformed cell line, and the industrially used methods include CHO dhfr (-) cell line, CHO K1, BHK cell line and NS0. (Ogata, et al., Appl. Microbiol. Biotechnol., 1993, 38 (4), 520-25; Kratje, et al., Biotechnol. Prog., 1994, 10 (4), 410-20; Peakman, et al., Hum.Antibodies Hybridomas, 1994, 5 (1-2), 65-74). Among these cell lines, CHO cell lines deficient in dihydrofolate reductase (DHFR) are the most widely used host cells for industrial mass production of target proteins using animal cells. The reasons why CHO cell lines lacking DHFR are industrially preferred are as follows. (1) Posttranslational modification of proteins, ie glycosylation or phosphorylation, is similar to human cells. (2) Suspension culture as well as adherent culture is possible. (3) Higher concentration cultures are possible in the serum-free medium compared to other cells. (4) Significantly lower target protein productivity compared to microorganisms can be increased using the dihydrofolate reductase / methoprexate (MHFR) / methotrexate (MTX) gene amplification system. (5) The safety has been verified and can be easily obtained from an oversight body such as the FDA. A recombinant CHO cell line producing a target protein is produced by transforming a CHO cell line deficient in this DHFR.

The main energy sources in culturing CHO cells are glucose and glutamine, and metabolism of CHO cells is largely related to glycolysis, TCA cycle, glutaminolysis and pentose phosphate. pathways). When glucose enters the cell, glycolysis produces pyruvate, the end product of glycolysis, and the pyruvate is converted to lactate by lactate dehydrogenase (LDH) or pyruvate decarboxylase (LDH). Enzymes such as pyruvate carboxylase (PC) and pyruvate dehydrogenase (PDH) can be introduced into the TCA cycle. Due to the characteristics of CHO cells that are continuously dividing, fast energy (ATP) rotation should be possible, and since the expression of PC and PDH in cells is less than that of LDH, most glucose accumulates in the medium through pyruvate and is inhibited by LDH. Is converted to lactic acid.

LDH is a reversible enzyme with subtype A and B. Since LDH-A, a form of A, catalyzes the reaction of generating lactic acid in pyruvic acid, inhibiting the expression of LDH-A can reduce the production of lactic acid. Expression of LDH-A was determined by homologous recombination (Chen et al., Biotechnol. Bioeng. 2001, 72 (1), 55-61), antisense mRNA (Jeong et al., Biochem. Biophys. Res. Commun. 2001). , 289 (5), 1141-9) and short interfering RNA (Kim and Lee, Appl. Microbiol. Biotechnol. 2007, 74 (1), 152-9).

Recombinant CHO cell lines and serum-free suspension culture are essential for the industrial production of target proteins using recombinant CHO cell lines. The reason for removing serum from the whole culture process is that there is a large amount of undefined animal protein in serum, so the use of serum-free medium can reduce the cost and effort of the downstream purification process. Supervisors such as this require the removal of serum from the entire process. However, in serum-free suspension culture of CHO cell line, apoptosis due to proliferation occurs early and progresses faster than in serum-containing cultures, resulting in reduced production of target protein. There are various causes of cell death, but when suspended in serum-free media, nutrients are depleted faster in culture compared to serum media, and waste products such as lactic acid and ammonia accumulate in the media to promote cell death.

In a previous study, we described a method for producing a target protein using CHO host cells transformed with anti-scheduled genes and lacking the DHFR gene (see Patent No. 10-0455016), and by expressing antisense LDH mRNA, LDH A method for producing a recombinant human EPO producing cell line with reduced expression of has been proposed (see Patent No. 10-2008-0101095). However, in the method of inhibiting the expression of LDH, the present inventors have prepared a host cell expressing short interfering RNA (siRNA) rather than an antisense LDH mRNA, thereby limiting the target protein to EPO and transforming a host of various target proteins. To be introduced into the cells. In addition, a method for producing a target protein using a CHO host cell in which an anti-stage gene and an LDH gene are simultaneously transformed has not been reported.

When the target protein is produced by recombinant CHO cells, the final concentration of the target protein is influenced by the concentration of viable cells producing the target protein and the nonprotein production rate (final protein concentration = cell concentration × nonprotein production rate). ). In conventional culture methodology, non-protein production rate is considered to be a factor that cannot be changed because it is regarded as the inherent property of cells. Therefore, in order to increase the final target protein concentration, it is necessary to increase the concentration of cells or increase The study was conducted to increase the cell culture period by inhibiting the cells.

Therefore, the present inventors introduced a vector expressing a shRNA that transforms a gene encoding Bcl2, an anti-digestive protein, and inhibits the expression of LDH-A in a cell line lacking DHFR, overexpressing Bcl2 and LDH-A. A low-expressing CHO DG44 cell line was prepared, and low expression of LDH-A, one of the enzymes involved in CHO cell metabolism, lowered the production of lactic acid, one of the culture wastes, and altered the overall metabolic flow of CHO cells. It was confirmed that the specific production rate of the protein increased, and at the same time, by overexpressing the anti-scheduled protein Bcl2, the cell concentration could be increased by increasing the incubation period, thereby confirming that the final protein concentration could be greatly improved. Completion of the present invention by revealing that it can produce a large amount of the protein of interest by reducing apoptosis and inhibit the production of lactic acid Respectively.

An object of the present invention is to introduce a novel CHO cell line transformed and lacking the DHFR gene, a method for producing the same, and a transformed CHO cell line by introducing a vector expressing a Bcl2 protein and a vector inhibiting the expression of an LDH-A enzyme. It is to provide a method for producing a target protein using.

In order to achieve the above object, the present invention is transformed to overexpress B-cell lymphoma 2 (Bcl2) protein, low expression of type A lactate dehydrogenase A (LDH-A) protein It provides a novel transformed CHO cell line lacking the DHFR gene.

The present invention also provides a method of producing a cell line that is overexpressed Bcl2 protein, transformed to low expression LDH-A protein, and lacks the DHFR gene.

In addition, the present invention provides a method of producing a target protein using the transformed CHO cell line after transforming the vector containing the gene encoding the target protein in the transformed CHO cell line.

The transformed CHO cell line of the present invention is inhibited by the cell proliferation due to overexpression of Bcl2 protein, the cell viability is excellent, not only can increase the production period of the target protein by increasing the culture period of the cell, but also inhibits the expression of the LDH-A enzyme When lactic acid production is lowered, the lactic acid accumulation in the culture medium decreases, and the flow of cell metabolism changes, thereby introducing a gene encoding the target protein, thereby increasing the specific production rate of the target protein. Since the resistance to the external environment is high, the integrity of the cell membrane can be maintained, and the amount of waste accumulated in the culture is reduced, so that the quality of the protein can be improved by producing the protein in a more comfortable culture environment. It can be usefully used for the preparation of. Therefore, the unit productivity of the cell line transformed to overexpress the Bcl2 protein of the present invention and low expression of the LDH-A protein, and the product of animal cells such as humanized antibodies and cytokines using the cell line preparation method and the cell line preparation method. And it can be usefully used for market development in related fields by improving the yield.

FIG. 1A is a diagram showing a genetic map of a vector (pcDNA3.1 / Zeo (+) / Bcl2) containing a Bcl2 gene.
Figure 1b is a diagram showing a genetic map of the vector (pSilencer3.1 / H1 hygro / LDHAsi) expressing shRNA for LDH-A.
2 is overexpression of Bcl2 protein and low expression of LDH-A in CHO DG44 host cells (Bcl2-LDHAsi) lacking the DHFR gene into which the vector containing the Bcl2 gene and the vector expressing shRNA against LDH-A were introduced. Western blot plot showing that:
Control (Null): CHO DG44 host cell into which a vector containing no Bcl2 gene and a vector containing no siRNA for LDH-A were introduced.
FIG. 3 is a graph showing cell growth curves and survival rates in batch culture of CHO DG44 host cells deficient in the DHFR gene into which the vector comprising the Bcl2 gene and the vector expressing shRNA for LDH-A were introduced and the control cells thereof. .
Figure 4 shows the change in the concentration of glucose and lactic acid in the medium during batch culture of the CHO DG44 host cell lacking the DHFR gene and the control cell for which the vector containing the Bcl2 gene and the vector expressing shRNA for LDH-A were introduced. The graph shown.
Figure 5a is a graph showing the cell growth curve and survival rate in the batch culture of the parent cell with the target protein introduced into the cell containing the vector containing the Bcl2 gene and the shRNA expressing the shRNA for LDH-A .
FIG. 5B shows the degree of apoptosis in the batch culture of parental cells into which a vector containing the Bcl2 gene and a vector expressing shRNAs for LDH-A were introduced and parent cells into which a target protein was introduced. Western blot plot of the expression of cleaved caspase-7.

Hereinafter, the present invention will be described in detail.

In the present invention, 'overexpression' generally means expressing at least two times or more of the amount of original cell expression.

In the present invention, 'low expression' typically means expressing at least 50% or less of the amount expressed by the original cell.

In the present invention, short hairpin RNA is processed into short interfering RNA (siRNA) by RNase III enzyme called Dicer in the cell into RNA of hairpin structure including the sequence of short interference RNA. As a result, siRNAs are generated from shRNAs expressed by shRNA vectors to inhibit the expression of LDH-A.

The present invention is a novel cell that overexpresses B-cell lymphoma 2 (Bcl2) protein, transforms to low expression of lactate dehydrogenase A (LDH-A) protein, and lacks the DHFR gene. One transformed CHO cell line is provided.

The cell line is preferably a cell line deposited with accession number KCTC11819BP, but is not limited thereto.

The cell line may be inhibited by cell scheduled death to prolong the incubation period of the cell, the lactic acid production is lowered, the concentration of lactic acid accumulated in the culture medium is preferably reduced, but is not limited thereto.

We prepared a CHO cell line (DG44 Bcl2-LDHAsi) that lacks the DHFR gene that overexpresses Bcl2 protein and low expresses LDH-A, and that cell death and lactic acid production by predose of the cell line are in serum-free suspension culture. Whether it was suppressed was examined. Specifically, control cells (DG44 Null) that do not express Bcl2 protein and low expression of LDH-A, or the DG44 Bcl2-LDHAsi cells were batch cultured, and then the survival rate of each cell was measured. As a result of batch culturing the cells in serum-free medium, control cell line rapidly decreased after 24 hours of inoculation and after 3 days of inoculation, and cell viability decreased. About 30% of cells died on the 5th day of culture and 50 on the 6th day of culture. More than% of the cells died, indicating a cell survival of about 45% (see Figure 3). In conclusion, in the case of DG44 Bcl2-LDHAsi cells, the cell culture period was extended by transformation.

The present inventors measured the concentrations of glucose and lactic acid in the serum-free medium using a metabolite analyzer (YSI7100, Yellow Spring Instrument, Yellow Springs, OH, USA), and as a result, the lactic acid specific production rate of DG44 Bcl2-LDHAsi cells was weak. 30% reduction was seen (see FIG. 4).

In order to confirm whether DG44 Bcl2-LDHAsi cells transformed to overexpress Bcl2 protein of the present invention prepared above and low expression of LDH-A can be used for production of a target protein, the DG44 Bcl2-LDHAsi cells The vector containing the gene of the protein of interest was transformed. As a result, cell populations overexpressing Bcl2 protein and low expression of LDH-A not only improved the growth and survival rate of the cells compared to the control group but also significantly increased the target protein production as the specific protein production rate increased. It was confirmed (see FIG. 5).

Thus, the DG44 Bcl2-LDHAsi cells are cell lines capable of floating culture in serum-free medium, which can prolong cell survival by inhibiting premature death in serum-free medium, and introduce a protein encoding a protein of interest. Since the protein of interest can be produced with high efficiency, it can be usefully used as a novel transformed CHO cell line which is transformed to overexpress Bcl2 protein and low express LDH-A protein and lacks DHFR gene.

The present invention also provides a method of producing a cell line that is overexpressed Bcl2 protein, transformed to low expression LDH-A protein, and lacks the DHFR gene.

The manufacturing method,

1) selecting a host cell overexpressing the Bcl2 protein after introducing a vector comprising the Bcl2 gene into a host cell deficient in the DHFR gene attached and cultured in a serum-containing medium;

2) After introducing a vector expressing a short hairpin RNA (shRNA) that inhibits the expression of LDH-A in a host cell overexpressing the Bcl2 protein selected in step 1), low expression of LDH-A protein Selecting a host cell; And

3) It is preferable to include a host cell over-expressing the Bcl2 protein selected in step 2) and low-expressing LDH-A protein to adapt to suspension culture in serum-free medium.

In the method, the host cell of step 1) is CHO, VERO, BHK, HeLa, Cv1, MDCK, 293, 3T3, myeloma, PC12 or WI38 are all available, preferably CHO cells, The CHO cell is more preferably a CHO cell deficient in the DHFR gene in order to amplify the target protein gene cloned in the vector into the DHFR / MTX system, and in particular, the CHO cell is most preferably a CHO DG44 cell. One is not limited thereto.

The CHO DG44 is commercially available as a cell line that does not express the DHFR protein involved in the synthesis of hypoxanthine and thymidine, which are essential components in cells (CHO DG44, Invitrogen, A11000-01). .

In the above method, the vector introduction into the host cell of steps 1) and 2) is performed by the method of transformation, electroporation, transduction, microinjection or ballistic introduction. All of these can be used, but is preferably a transformation method, but is not limited thereto.

The liposomes mixed into the vector for transformation are at least one selected from the group consisting of lipofectin, lipofectamine, oligofectin, dotap, and superperfect. It can be used, it is preferred to use lipofectamine, but is not limited thereto.

It is preferable that the host cell not only overexpresses Bcl2 protein, which is an anti-stage protein, by the method of steps 1) and 2) but also decreases the expression of LDH-A.

In order to prepare CHO cell lines transformed with Bcl2 and LDH-A genes and lacking DHFR, we used CHO DG44 host cells with 1X hypoxanthine / thymidine supplement (HT supplement, Gibco) and 7% (v). / v) adherent culture in IMDM medium containing dialyzed fetal bovine serum. After introducing a Bcl2 overexpression vector (pcDNA3.1 / zeo (+) / Bcl2) (see FIG. 1A) into the host cell in adherent culture and selecting cells resistant to zeocin antibiotics, Western blot The cell lines overexpressing Bcl2 protein were selected by the blot method. In addition, a vector expressing shRNA that inhibits the expression of LDH-A in a DHFR deficient CHO cell line overexpressing the selected Bcl2 protein to select a cell line that inhibits the production of lactic acid (pSilencer3.1 H1-hygro_LDHAsi) (FIG. 1B). Cells were selected for resistance to hygromycin antibiotics, and then, Western blot methods were used to select low-expressing LDH-A cells. As a result, a CHO cell line (DG44 Bcl2-LDHAsi) lacking the DHFR gene overexpressing Bcl2 protein and low expressing LDH-A was prepared through two cell selection processes. The DG44 Bcl2-LDHAsi cell line prepared as described above was deposited on the microbial resource center of the Korea Research Institute of Bioscience and Biotechnology on December 3, 2010 [Accession No .: KCTC 11819BP (DG44 Bcl2-LDHAsi (CHO cell line)].

The present inventors carried out suspension culture in Hyclone SFM4CHO ^TM medium (Hyclone, Logan, UT, USA), a CHO cell line that lacks the DHFR gene that overexpresses the Bcl2 protein prepared above and low expresses LDH-A. It was. When the cells reached the logarithmic growth phase, they were passaged in the same culture environment and adapted to suspension culture until the growth rate of the cells recovered the growth rate of the cells in the attached culture.

The DG44 Bcl2-LDHAsi cells prepared as described above are inhibited in premature death due to the overexpression of the anti-scheduled protein Bcl2, which increases cell viability and accumulates in the culture medium due to the low expression of lactic acid producing enzyme LDH-A. The production of lactic acid, which is one of the waste products, was suppressed to inhibit the accumulation of lactic acid. By manipulating genes related to cell survival and cell metabolism, the two traits were simultaneously converted, and this transformation changed the flow of cell metabolism to increase the productivity of the target protein while increasing the cell survival rate. Improved.

Therefore, by transforming to overexpress Bcl2 protein and low express LDH-A protein, it can be usefully used for the production of cell lines with improved productivity of the desired protein.

In addition, the present invention provides a method for producing a target protein using the transformed CHO cell line after transforming a vector comprising a gene encoding the target protein in the transformed CHO cell line.

The method comprises:

1) preparing a transformed CHO cell line to express the target protein by introducing a vector containing a gene encoding the target protein into the transformed CHO cell line;

2) culturing the cell line prepared in step 1); And

3) preferably, but not limited to, separating and purifying the target protein produced in the cell line of step 2).

The method for producing the target protein can be obtained by transforming the transformed CHO cell line with a vector comprising a gene encoding the target protein.

In the method, the target protein of step 1) is soluble TNF receptor, soluble IL-4 receptor, soluble IL-1 type II receptor, soluble Flt3 ligand, soluble CD40 ligand, CD39, CD30, CD27, TEK / Ork, IL- 15, Soluble IL-15 Receptor, Ox 40, GM-CSF, RANKL, RANK, TRAIL, Soluble TRAIL Receptor, Tissue plasminogen activator, Factor Ⅷ, Factor Ⅸ, Apofat Apolipoprotein E, apolipoprotein AI, IL-2 receptor, IL-2 antagonist, alpha-1 antitrypsin, calcitonin, growth hormone Insulin, insulin, insulin-like growth factors, insulin-like growth factors, parathyroid hormone, interferons, superoxide dismutase , Glucagon, erythropoeitin, antibodies, Group consisting of glucocerebrosidase, Fc-fusion protein, globins, nerve growth factors, interleukins, colony stimulating factors It is preferably selected from, and more preferably is an Fc-fusion protein, but is not limited thereto.

In order to confirm whether the CHO cell line transformed to overexpress the Bcl2 protein prepared above and low expression of LDH-A can be used for the production of the target protein, the transformed CHO cell line was 7% (v / The cells were inoculated with IMDM medium containing dFBS and hypoxanthine / thymidine supplement of v) and cultured, and then the vector containing the gene of the desired protein was transformed into the cells. Then, a cell line into which the vector containing the gene of the protein of interest is introduced is added to the medium, and a cell line (transformed cell line) resistant to the cell is selected by adding a selection antibiotic to the medium. pooling and screening or individual clones. In the present embodiment, the cells producing the protein of interest were selected with a collection pool to exclude the effects of clone variation that may occur when the clones were selected. The cells established in this way are called parental cells. The parent cells were subjected to gene amplification of the target protein for 2 to 3 weeks in IMDM medium containing 7% (v / v) dFBS containing methodotrexate (MTX). Through batch culture of cells, the unit productivity of the target protein was determined to determine whether the production of the protein was increased. The control group (Protein Null) which introduced the gene encoding the Fc-fusion protein into the CHO DG44 host cell which does not overexpress the Bcl2 protein and does not low LDH-A, and overexpresses the Bcl2 protein and low expresses LDH-A. After cell culture (Protein Bcl2-LDHAsi) in which a gene encoding an Fc-fusion protein was introduced into CHO DG44 host cells was attached and cultured in a serum-containing medium, the growth, viability and production of Fc-fusion protein were measured. As a result, the protein Bcl2-LDHAsi cells showed both reduced glucose specific consumption rate and lactic acid specific production rate as compared to the control group (see Table 2 and FIG. 4), as well as improved cell growth and survival rate as well as no production of target protein. As the rate was increased, it was confirmed that the yield of the target protein was significantly increased (see Table 1 and FIG. 5).

From the above results, the DG44 Bcl2-LDHAsi host cell line of the present invention is a cell line capable of floating culture in a serum-free medium, which can prolong cell survival by inhibiting precocious death in a serum-free medium, and encodes a gene of interest protein. It was confirmed that the present invention can be used as a highly productive protein producing cell line.

Thus, novel transformed CHO cell lines transformed to overexpress Bcl2 protein and low express LDH-A protein and lack the DHFR gene can be usefully used for the production of the protein of interest.

Hereinafter, the present invention will be described in detail with reference to the following examples.

However, the following Examples illustrate the present invention in detail, and the content of the present invention is not limited by the following Examples.

human's Bcl2  Overexpress proteins and intracellular LDH -A Underexpressed DHFR Deficient CHO  Manufacture of cell lines

To prepare a DHFR deficient CHO cell line that overexpresses human Bcl2 protein, first a DHRF deficient CHO DG44 host cell (Invitrogen, A11000-01) was added with a 1 × hypoxanthine / thymidine supplement (HT supplement, Adhesion cultures were carried out in IMDM (Iscov's Modified Dulbecco's Medium, Gibco) medium containing Gibco, Grand Island, NY, USA) and 7% (v / v) dFBS. A pcDNA3.1 / Zeo (+) / Bcl2 (FIG. 1A) vector, which is a vector encoding a gene expressing Bcl2, was introduced into a CHO DG44 host cell cultured with attachment using lipofectamine (lipofectamine 2000, Invitrogen). 2-3 days after inoculating 1 × 10 ⁶ cells in a T-25 flask in IMDM medium containing 5 ml of 1 × hypoxanthine / thymidine supplement and 7% dFBS, the cultured cells were placed in the flask. After at least 90%, 8 μg of DNA and 20 μl of lipofectamine complex were added to OptiMEM (Gibco) medium, followed by incubation for 24 hours. Exactly 24 hours after the introduction of the vector, cells resistant to antibiotics were selected by incubating in 96 well plates for 2-3 weeks with 300 μg / ml zeocin antibiotic. After screening cells resistant to zeocin antibiotics, a cell line (DG44 Bcl-2) overexpressing Bcl2 protein was selected through Western blot. In order to suppress LDH-A expression of the cell line overexpressing the selected Bcl2 protein, the pSilencer3.1 / H1 hygro / LDHAsiRNA (FIG. 1B) vector containing siRNA sequences that inhibit the expression of LDH-A was attached and cultured. DG44 Bcl2 cell line was introduced using lipofectamine. Exactly 24 hours after the vector was introduced, cells resistant to antibiotics were selected by incubating in a 96 well plate for 2-3 weeks with 500 μg / ml of hygromycin antibiotic. After selecting cells resistant to hygromycin antibiotics, Western blot was used to select a cell line (DG44 Bcl2-LDHAsi) low expression LDH-A. At this time, a vector without a gene sequence encoding Bcl2 protein (pcDNA3.1 / Zeo (+)) and a vector without a siRNA sequence expressing low LDHA (pSilencer / H1 hygro / scrambledsiRNA) were introduced to zeocin and hygromycin. Cells selected in the culture medium containing antibiotics were used as control cells (DG44 Null).

As a result, it was confirmed that the DG44 Bcl2-LDHAsi cell line stably expresses 26 kDa of Bcl2 and that 37 kDa of LDH-A expression is significantly lower than that of the control cells (FIG. 2). Therefore, the DG44 Bcl2-LDHAsi cell line was deposited on the microbial resource center of the Korea Research Institute of Bioscience and Biotechnology on December 03, 2010 (Accession No .: KCTC 11819BP, DG44 Bcl2-LDHAsi (CHO cell line)].

Bcl2 Overexpress LDH -A Underexpressed DHFR Deficient CHO  Measurement of Cell Growth and Cell Metabolites Concentration in Cell Lines

The serum-free suspension culture was applied to the adherent cultured CHO DG44 Bcl2-LDHAsi cell line obtained in Example 1 to measure cell growth and concentration of cell metabolites in the medium. Attached cultured cells were cultured with HyClone SFM4CHO ^™ (Hyclone, Logan, UT, USA) medium containing 1 × Hypoxanthine / Thymidine Supplement, 300 μg / ml Zeocin, 500 μg / ml Hygromycin, 4 mM Glutamine 50 ml of the seed was inoculated at an initial concentration of 5 × 10 ⁵ cells / ml and placed in an Erlenmeyer flask to carry out suspension culture. Three days after the start of the culture, when the cells reached the exponential phase, they were passaged in the same culture environment. At 3 days after passage, the cell concentration reached 1 × 10 ⁶ cells / ml and the initial cell concentration was lowered to 3 × 10 ⁵ cells / ml, followed by suspension culture. Upon reaching 10 ⁶ cells / ml, the initial cell concentration was lowered to 2 × 10 ⁵ cells / ml to perform suspension culture to adapt the cells to serum free suspension culture. Thereafter, batch culture of DG44 Bcl2-LDHAsi and control cells (DG44 Null) adapted to serum-free suspension culture was used to measure cell growth and cellular metabolite concentration.

As a result, similar growth rate and survival rate were observed until the 4th day of the batch culture, but the survival rate of the control cells rapidly decreased thereafter, and the survival rate dropped to about 30% on the 7th day. As a result of calculating the cumulative cell number obtained during the incubation period, the cells overexpressing Bcl2 and low expressing LDH-A were more than twice as many as the control cells (FIG. 3). As a result of measuring the concentrations of glucose and lactic acid in the medium during the batch culture, the lactic acid production rate of the cells overexpressing Bcl2 and low expression of LDH-A was about 50% slower than that of the control cells, and the final maximum lactic acid concentration produced was It was lower than about 20 mM of control cells as measured at 17 mM (FIG. 4). As a result, by overexpressing Bcl2 and low expression of LDH-A, CHO cells could more than double the total number of cells proliferated while reducing the amount of waste in the medium.

Bcl2 Overexpress LDH -A Underexpressed DHFR Deficient CHO  Production of target protein using cell line

In order to confirm whether the CHO host cell line deficient in DHFR, which overexpresses Bcl2 and low in LDH-A, obtained in Example 1, can be used for production of a target protein, the following experiment was performed. . First, cells were seeded at a concentration of 2 × 10 ⁵ cells / ml in a T-25 flask with 5 ml of IMDM medium containing 1 × hypoxanthine / thymidine supplement and 7% (v / v) of dFBS. . After 2 to 3 days, when the cultured cells are filled in the flask by 90% or more, the gene of the target protein is added to the DHFR-deficient CHO cell line that overexpresses Bcl2 prepared in Example 1 and low expresses LDH-A. The vector containing was mixed with liposomes and introduced into the cells. Exactly 24 hours later cells were seeded at a concentration of 1 × 10 ⁵ cells / ml in a T-75 flask with 15 ml of IMDM medium containing 7% (v / v) dFBS without hypoxanthine / thymidine The transformed cell lines were then selected by adding antibiotics. In selecting a cell line (transformed cell line) resistant to antibiotics, either a pool of resistant cells can be collected or selected by individual clones, and here, a pool of pools can be used. Collected and selected. The medium was changed every 5 to 7 days while checking the state of the cells under a microscope. After about 2 weeks, when the cells were filled in the T-75 flask, the cells were treated with trypsin (Gibco) in the T-75 flask. After the recovery, the cells were counted, and the cells were inoculated at a concentration of 1 × 10 ⁵ cells / ml in a T-25 flask containing 5 ml of culture medium, and then batch culture was performed to confirm the productivity of the target protein. The cells established in this way are called parental cells. At this time, the medium was recovered and subjected to ELISA analysis for the target protein. The target protein unit productivity of the parent cells was measured using ELISA and the number of cells collected daily. In order to obtain more productive cells of the target protein, genes of the target protein were amplified by culturing the parent cells in a medium containing methodotrexate. Specifically, parental cells were seeded in a T-75 flask at a concentration of 1 × 10 ⁴ cells / ml, followed by 2-3 weeks in IMDM medium containing 20 nM mesotrexate and 7% (v / v) of dFBS. Gene amplification of the protein was performed and after 2-3 weeks the unit productivity of the target protein of the selected cells was measured. In addition, the gene amplified cells in a medium containing 20 nM of mesotrexate is amplified once more in a medium containing 80 nM of mesotrexate in the same manner as described above through the batch culture of the selected cells after 2-3 weeks Unit productivity of the desired protein was measured. As described above, a cell line deficient in DHFR that does not overexpress Bcl2, a control cell line, and low LDH-A, was also produced. Cell growth of cell lines (Protein Bcl2-LDHAsi) and control cells (Protein Null) overexpressing Bcl2 producing the target protein prepared above and low LDH-A, unit productivity of the target protein and cell metabolites in the culture medium The concentration of was determined to determine the effects of overexpression of Bcl2 and low expression of LDH-A.

As a result, the control cells rapidly reduced cell viability after 5 days of batch culture and dropped to 50% or less on day 6 of culture, while overexpressing Bcl2 and low expressing LDH-A and producing a target protein. Showed a high survival rate of about 70% even on the 8th day of culture, and the survival rate dropped to 50% or less on the 9th day of culture (FIG. 5A). Western blot method was used to measure the expression level of cleaved caspase-7 and to confirm cell death during batch culture.By using the results, the cells produced to overexpress Bcl2 and low LDH-A were cultured. On day 9, they expressed significantly less cleaved caspase-7 compared to control cells (FIG. 5B). As a result of calculating the specific consumption rate of glucose and lactic acid by measuring the concentrations of glucose and lactic acid in the medium during batch culture, the genes amplified compared to the control cells in cells designed to overexpress Bcl2 and low LDH-A Depending on the degree, glucose specific consumption rate decreased to about 35-50% of the control group, and the specific production rate of lactic acid decreased to about 20-50% level (Table 2). Therefore, in the case of cells that are overexpressed with Bcl2 and low in LDH-A, fed-batch and perfusion cultures, instead of batch culture, consume less glucose and produce less lactic acid. It can be seen that the increase in cell resistance due to the overexpression of Bcl2 can further extend the cell culture period. In addition, since the unit productivity of the target protein of the cell line produced through genetic manipulation of the Bcl2 and LDH-A genes is also superior to that of the control cells, the unit productivity of the target protein is increased in addition to the extension of the cell culture period, thereby increasing the mass production of the target protein. It can be seen that the host cell lacking DHFR that overexpresses and low expresses LDH-A can be effectively used (Table 1). In particular, since cells which are designed to overexpress Bcl2 and low expression of LDH-A are host cells, they can be used for production of a desired target protein depending on which target protein gene containing a vector is introduced into the host cell.

Target _protein unit productivity ( q _protein ) of control cells and cells designed to overexpress Bcl2 and low expression of LDH-A q _protein (μg / 10 ⁶ cells / day) Null Bcl2-LDHAsi Parent cell 2.12 ± 0.03 1.01 ± 0.09 Cells Amplified with 20nM MTX 16.05 ± 0.68 20.97 ± 0.25 Cells amplified with 80 nM MTX 15.94 ± 0.83 30.82 ± 1.71

Glucose specific consumption rate ( q Glc (μmol / 10 ⁶ cells / day)), lactic acid specific production rate ( q Lac (μmol / 10 ⁶ cells /) of control cells and cells designed to overexpress Bcl2 and low expression of LDH-A day) and lactic acid production per glucose ( Y _{Lac / Glc} (mol / mol)) Parent cell q Glc
(μmol / 10 ⁶ cells / day) ratio q Lac
(μmol / 10 ⁶ cells / day) ratio Y _{Lac / Glc} (mol / mol) ratio Null 4.19 ± 0.16 One 6.77 ± 0.33 One 1.62 ± 0.02 One Bcl2-LDHAsi 3.09 ± 0.18 0.74 3.24 ± 0.21 0.48 1.05 ± 0.01 0.65 Cells Amplified with 20nM MTX q Glc
(μmol / 10 ⁶ cells / day) ratio q Lac
(μmol / 10 ⁶ cells / day) ratio Y _{Lac / Glc} (mol / mol) ratio Null 4.67 ± 0.46 One 7.40 ± 0.28 One 1.59 ± 0.10 One Bcl2-LDHAsi 2.49 ± 0.04 0.53 1.53 ± 0.08 0.21 0.62 ± 0.04 0.39 Cells amplified with 80 nM MTX q Glc
(μmol / 10 ⁶ cells / day) ratio q Lac
(μmol / 10 ⁶ cells / day) ratio Y _{Lac / Glc} (mol / mol) ratio Null 4.61 ± 0.23 One 8.64 ± 0.25 One 1.88 ± 0.15 One Bcl2-LDHAsi 2.64 ± 0.26 0.57 1.84 ± 0.11 0.21 0.70 ± 0.11 0.37

Korea Biotechnology Research Institute KCTC11819BP 20101203

Claims (8)

CHO cell line deposited with accession number KCTC11819BP.
delete 1) introducing a vector comprising the Bcl2 gene into a host cell deficient in the DHFR gene attached and cultured in a serum-containing medium, and then selecting a host cell overexpressing the Bcl2 protein;
2) After introducing a vector expressing a short hairpin RNA (shRNA) that inhibits the expression of LDH-A in a host cell overexpressing the Bcl2 protein selected in step 1), low expression of LDH-A protein Selecting a host cell; And
3) adapting the host cell overexpressing the Bcl2 protein selected in step 2) and low expressing the LDH-A protein to suspension culture in serum-free medium,
Wherein said host cell is a CHO dhfr (-) cell or a CHO DG44 cell, wherein said host cell is transformed to overexpress Bcl2 protein and low expresses LDH-A protein and is deficient in DHFR gene.
delete delete delete 1) preparing a CHO cell line transformed to express a target protein by introducing a vector comprising a gene encoding the target protein into the CHO cell line of claim 1;
2) culturing the cell line prepared in step 1); And
3) Isolating and purifying the target protein produced in the cell line of step 2).
The method of claim 7, wherein the target protein is soluble TNF receptor, soluble IL-4 receptor, soluble IL-1 type II receptor, soluble Flt3 ligand, soluble CD40 ligand, CD39, CD30, CD27, TEK / Ork, IL-15, Soluble IL-15 Receptor, Ox 40, GM-CSF, RANKL, RANK, TRAIL, Soluble TRAIL Receptor, Tissue plasminogen activator, Factor Ⅷ, Factor Ⅸ, Apolipoprotein ( apolipoprotein E, apolipoprotein A-I, IL-2 receptor, IL-2 antagonist, alpha-1 antitrypsin, calcitonin, growth hormone Insulin, insulin, insulin-like growth factors, insulin-like growth factors, parathyroid hormone, interferons, superoxide dismutase , Glucagon, erythropoeitin, antibody, glue From the group consisting of cerebrosidase, Fc-fusion protein, globins, nerve growth factors, interleukins, colony stimulating factor Method for producing a target protein, characterized in that any one selected.

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