KR100679112B1 - Method for Animal Cell Culture - Google Patents

Abstract

The present invention provides a method for culturing animal cells producing a recombinant protein, comprising: preparing a hydrolysis product produced by hydrolyzing a soybean or yeast extract protein with an acid or an enzyme; Culturing the cells in serum-free medium while maintaining an oxygen concentration of at least 50% and a pH of 7.2; And culturing the cells after adding the hydrolysis product in an amount of 3 g / L to 20 g / L when the cell concentration reaches 1.5 × 10 ⁶ cell lines / ml. . In addition, the method for culturing animal cells producing the recombinant protein of the present invention is a hydrolyzate produced by hydrolyzing soybean or yeast extract protein with acid or enzyme, 3 g / L to 20 g / Continuously culturing the cells in the L-containing serum free medium may be further included. According to the present invention, a recombinant protein can be produced in large quantities by increasing the rate of protein degradation by at least five times while alleviating cell death.

Animal cells, cell culture, recombinant protein, serum-free medium

Description

Method for Animal Cell Culture

1 is a graph showing cell growth and survival rate of the medium to which the protein hydrolyzate is added.

Figure 2 is a graph showing the production concentration of protein in the medium to which the protein hydrolyzate is added.

3 is a graph showing cell growth and survival rate in a batch culture using a medium to which a protein hydrolyzate is added.

Figure 4 is a graph showing the protein production concentration in batch culture using a medium to which the protein hydrolyzate is added.

5 is a graph showing cell growth and survival rate in continuous culture using a medium to which a protein hydrolyzate is added.

Figure 6 is a graph showing the protein production concentration during continuous culture using a medium to which a protein hydrolyzate is added.

The present invention relates to a method for culturing animal cells producing a recombinant protein, specifically, using a serum-free medium as a cell growth medium, after sufficiently growing the cells, protein hydrolysates [soybean or yeast ( yeast) can be increased by at least 5 times the rate of secretion of the protein by culturing with the addition of the hydrolyzate produced by hydrolysis of the extracted protein with an acid or an enzyme to reduce cell death.

To date, many methods have been used for culturing animal cells to increase protein production, and the methods can be divided into two categories. The first is to create a highly productive animal cell line. When using a eukaryotic cell such as an animal cell as a host cell, there is a disadvantage in that the expression amount of the recombinant protein is less than that of using recombinant E. coli or recombinant yeast as the host cell. To overcome this disadvantage, gene amplification using selection marker genes such as Dihydrofolate Reductase (dhfr), Glutamine Synthetase (GS), and Adenine deaminase The method was used. Gene amplification increases the expression level of recombinant proteins by greatly increasing the number of genes in the cell, but when gene amplification is excessive, they are expressed but secreted to the outside of the cell at a slower rate, resulting in complete post translational modification. There was an unfavorable disadvantage (Martin Scholer et. Al. Biotechnology and Bioengineering, 547-559, Vo. 53, No. 6, 1997).

The second method is an optimization of culture conditions, and various culture variables have been studied for this purpose. The primary goal of optimizing the culture conditions is to achieve the highest possible concentration of cells, and the biggest problem in achieving high concentrations of cell culture is the depletion of nutrients necessary for cell growth, which is even more true for cultures using serum-free medium. It is sensitive. The best method of supplying nutrients necessary for cell growth and achieving high concentration culture is the continuous culture method, in which the spin-filter as the cell holding device for continuous culture is 2 × 10 ⁷ cells / High concentration cultures of more than ml are known (Reuveny et. al. Journal of Immunological Methods, 86: 61-69, 1985).

After culturing the cells at the highest concentrations, the culture parameters are adjusted to increase the protein production rate of the cells. Generally, a method of controlling the culture temperature, pH, dissolved oxygen and osmotic pressure is used, and a method of adding a new additive to the culture medium is used. These methods utilize the phenomenon of increasing the rate of protein secretion while slowing cell growth by increasing the proportion of cells present in the G1 state in the cell cycle (Kaufmann H et al. Biotechnology and Bioengineering, 63). : 573-582, (1999), Marc Cherlet et. Al. Cytotechnology 29: 71-84, (1999). Sodium butyrate is one of the additives used in the culture medium. To date, sodium butyrate is the most used to increase the expression and secretion of proteins in animal cell culture. Sodium butyrate is absorbed into the cell nucleus, weakening the binding of DNA and histone proteins, increasing the acetylation of histone proteins and reducing phosphorylation, resulting in cells staying in the G1 state. It is known to increase the rate of recombinant protein production in cells (Marc Cherlet et. Al. Cytotechnology 32: 17-29, (2000)).

However, the addition of sodium butyrate in culture can increase the expression and secretion of recombinant proteins, but the addition of sodium butyrate stops cell growth and leads to cell death (Palermo. DP et. Al.). , J. of Biotechnology, 19 35-48 (1991), Chotigeat W., et. Al., Cytotechnology, 15, 217-221 (1994)). As the cell death occurs, the survival rate of the cells is sharply lowered, thereby shortening the production culture time. As a result, the total yield of the recombinant protein is decreased during continuous culture.

Protein hydrolyzate is an acid or enzymatic hydrolysis of soybean, wheat or yeast extract protein. It is used in food manufacturing (Korean Patent Application No. 1999-0031026) or when fermenting microorganisms (Korean Patent Application No. 1994). -0011070) has been used as an important nitrogen source. Recombinant protein production using animal cells has been used in conjunction with other growth factors such as insulin to replace fetal bovine serum (WO 96/26266). It has been used as a component (WO 2004/005493). However, previous studies have focused mainly on cell growth and, even when used for the production of recombinant proteins, their production rates were similar or equivalent to those of serum use (Xuejun Gu et. Al. Biotechnology and Bioengineering, 56: 353). -360, (1997)).

Accordingly, the present inventors have endeavored to develop an animal cell culture method capable of increasing the production time of recombinant protein by inhibiting cell death as much as possible and increasing the recombinant protein production rate of the cell, thereby producing the recombinant protein of the present invention. A method of culturing animal cells has been developed.

The present invention comprises the steps of growing cells in a serum-free medium; And adding a protein hydrolyzate (a hydrolyzate produced by hydrolyzing a soybean or yeast extract protein with an acid or an enzyme) to the serum-free medium. An object of the present invention is to provide a culture method.

In addition, the present invention comprises the steps of growing cells in a serum-free medium; And replacing the serum-free medium with a serum-free medium for production containing a protein hydrolyzate (a hydrolyzate produced by hydrolyzing a soybean or yeast extract protein with an acid or an enzyme). It is an object of the present invention to provide a method for culturing animal cells producing a recombinant protein.

The present invention relates to a method of culturing animal cells producing recombinant protein.

In one embodiment, the present invention provides sufficient growth of cells using a serum-free medium as a cell growth medium, followed by hydrolysis of protein hydrolyzate (soybean or yeast extract protein with acid or enzyme). Batch culture method of a continuous stirred tank reactor in which the resulting hydrolysis product] is added to the medium. Specifically, the present invention provides a method for culturing animal cells producing recombinant proteins, comprising: preparing a hydrolyzate produced by hydrolyzing a soybean or yeast extracted protein with an acid or an enzyme; Culturing the cells in serum-free medium while maintaining an oxygen concentration of at least 50% and a pH of 7.2; And culturing the cells after adding the hydrolysis product in an amount of 3 g / L to 20 g / L when the cell concentration reaches 1.5 × 10 ⁶ cell lines / ml. . The method comprises the steps of continuously culturing the cells in serum-free medium containing 3 g / L to 20 g / L of the hydrolysis product produced by hydrolyzing the soybean or yeast extract protein with acid or enzyme It may further include.

In another embodiment, the present invention provides sufficient growth of cells using a serum-free medium as a cell growth medium, followed by hydrolysis of a protein hydrolyzate (soybean or yeast extract protein with acid or enzyme). It is a continuous culture (Continuation culture) method of a continuous stirred tank reactor (exchanged with the produced hydrolysis product) serum-free medium containing.

Animal cells of the present invention includes all animal cells that can be used as genetically engineered animal cells for foreign protein production, the animal cells used in the animal cell culture method of the present invention Chinese Hamster Ovary (Chinese Hamster Ovary , Hereinafter abbreviated as 'CHO'.

Recombinant protein of the present invention is any protein that can be used in human therapeutic and diagnostic medicine, preferably a protein secreted with a sugar chain may be used, more preferably interferon-beta (eryfero-beta), erythropoietin ( erithropoietin, EPO), or coagulation factor 8 (antihemophilic factor, Factor VII) and the like can be preferably used.

Serum-free production medium of the present invention, including CHO-SFM-SGM1 (Korean Patent Application No. 10-2003-0099633), such as CHO PF medium of Sigma (USA), SFM4CHO medium of Hyclone (Hyclone, USA), etc. Serum-free medium already known commercially can be used.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the examples.

Examples 1 to 3 and Comparative Examples 1 to 3. Measurement of cell concentration, viability, secretion of recombinant protein

CHO cells transformed to produce interferon beta were inoculated in 175 mm ² T-flask using a medium containing 10% fetal bovine serum (FBS, Hyclone) in α-MEM (Gibco) medium, 37 ° C., Incubated for about 2 days in a 5% CO ₂ incubator. When the cells have grown sufficiently to reach about 2 × 10 ⁷ cells, the culture medium is removed, washed with PBS solution, detached cells attached to the flask with 0.1% trypsin solution, and inoculated into an Erlenmeyer flask containing CHO-SFM-SGM1 medium. Serum-free suspension culture was performed. Inoculated with about ⁵ × 10 ⁵ cell lines / ml and incubated for 2 days. After incubation for 2 days, the cell concentration reached 1.5X10 ⁶ cell line / ml or more, and the culture medium was added to the concentration of 5 g / L of proteolytic material for 2 days (Example 1), and nothing was added (comparatively). Example 1-1) The same procedure was followed except adding 1 mM sodium butyrate (Comparative Example 1-2).

CHO cells transformed to produce erythropoietin and coagulation factor 8 were also cultured in the same manner as described above (Examples 2 and 3 Comparative Examples 2-1, 2-2 and 3-1, 3-2).

When nothing was added to the culture medium (Example 1) to which a protein hydrolyzate (hydrolyzate produced by hydrolyzing soybean or yeast extract protein with acid or enzyme) was added (Comparative Example 1) Similar to -1), cell growth was observed, and cell viability was maintained until the end of the culture, whereas when sodium butyrate was added (Comparative Example 1-2), cell growth decreased and survival rate gradually decreased ( 1).

In the case of using the cell line producing interferon beta, the rate of protein secretion was increased by about 6 times compared to the case in which nothing was added, and about 2 times as compared to the case of adding sodium butyrate (Table 1 and FIG. 2). The cell line producing erythropoietin was increased about 6 times in protein production compared to the case in which nothing was added, and about 3 times as compared to the case in which sodium butyrate was added. In addition, it increased about 5 times in protein production compared to the case of adding nothing using the cell line producing the coagulation factor 8, and increased about 2.5 times compared to the case of adding sodium butyrate (Table 1).

Cell concentration (10 ⁶ / ml) Protein concentration Example 1 3.05 14.9 (ug / ml) Example 2 3.20 76.5 (ug / ml) Example 3 3.11 12.8 (IU / ml) Comparative Example 1-1 2.96 2.3 (ug / ml) Comparative Example 1-2 1.65 7.2 (ug / ml) Comparative Example 2-1 2.75 12.7 (ug / ml) Comparative Example 2-2 1.89 26.1 (ug / ml) Comparative Example 3-1 2.89 2.4 (IU / ml) Comparative Example 3-2 1.91 5.2 (IU / ml)

Example 4 Batch Culture with Incubator

Batch culture was performed in a 7.5 liter incubator (Celligen plus, NBS). CHO cells transformed to produce interferon beta were inoculated in T-flask using a medium containing 10% fetal bovine serum (FBS, Hyclone) in α-MEM (Gibco) medium, 37 ° C., 5% Incubation was performed for about 2 days in a CO ₂ incubator. The cell grows sufficiently and the number of cells about 2X10 ⁷ . When T-175 was reached, the culture medium was removed, washed with PBS solution, and the cells attached to the flask with 0.1% trypsin solution were removed and placed in a 250 ml spinner flask containing 100 ml of CHO-SFM-SGM1 (-) medium. Serum-free suspension culture was performed by inoculation.

Two days of inoculation with about ⁵ × 10 ⁵ cell lines / ml was incubated and then transferred to a 1 liter spinner flask containing 400 ml of CHO-SFM-SGM1 medium at a concentration of about 1.5 × 10 ⁶ cell lines / ml. Two 1-liter flasks were incubated for 3 days, and the entire amount of the culture solution was transferred to an incubator containing 2.5 liters of CHO-SFM-SGM1 medium at a concentration of 1.2 × 10 ⁶ cell lines / ml. Cultivation was carried out at 37 ℃ and maintained at a dissolved oxygen concentration of more than 50% by the injection of air and oxygen, and cultured while maintaining the pH at 7.2 by injection of sodium bicarbonate and carbon dioxide gas. When the cell concentration reached 1.5X10 ⁶ cell line / ml on the 3rd day of culture, the protein hydrolyzate (hydrolyzate produced by hydrolyzing soybean or yeast extract protein with acid or enzyme) was injected. Incubated to day 1 (Example 4), cultured to the end with CHO-SFM-SGM1 medium (Comparative Example 4-1) or sodium butyrate was added on day 3 of culture (Comparative Example 4-2).

Cell growth and viability were similar in the case of addition of the protein-digested substance (hydrolyzate produced by hydrolyzing soybean or yeast extract protein with acid or enzyme). When sodium butyrate was added, cell growth was decreased and viability was decreased (FIG. 3). Also, when protein hydrolysates (hydrolysates produced by hydrolyzing soybean or yeast extract proteins with acids or enzymes) are added, they are larger from the point of addition than when nothing is added. As a result, the protein production concentration was increased, resulting in an increase of about 10 times, and about 2 times as compared to the case of adding sodium butyrate (FIG. 4).

Example 5 Continuous Culture with Incubator

Continuous culture was performed in a 7.5 liter incubator (Celligen plus, NBS) equipped with a spin-filter having a mesh size of 15 μm. The batch culture was carried out by the method described in Example 4, and the medium exchange type continuous culture was performed when the cell concentration reached 1.5 × 10 ⁶ cell lines / ml. The first cell growth exchange medium was either CHO-SFM-SGM1 medium or commercially available medium, and the cell concentration was increased to ⁷ × 10 ⁶ cell lines / ml by changing the medium exchange rate at a rate of 0.5 to 4 times the volume of the culture medium per day. Subsequently, the medium was replaced with a production medium, followed by continuous culture with medium exchange to produce protein. Production medium includes CHO-SFM-SGM1 medium containing 5 g / L of protein hydrolyzate (hydrolyzate produced by hydrolyzing soybean or yeast extract protein with acid or enzyme). Example 5) Or no medium was added (Comparative Example 5-1) or 1 mM of sodium butyrate was added (Comparative Example 5-2).

CHO-SFM-SGM1 medium containing 5 g / L of protein hydrolysates (hydrolysates produced by hydrolyzing soybean or yeast extract proteins with acids or enzymes) is about the maximum cell concentration. Growth was continued in culture up to 20 × 10 ⁶ cell lines / ml. In other words, it was about 10% higher than no addition to the production medium, and about 2.5 times higher than the addition of sodium butyrate. In addition, when sodium butyrate was added, the survival rate dropped rapidly after reaching ⁸ × 10 ⁶ cell lines / ml, whereas the proteolytic material (soybean or yeast extract protein was produced by hydrolysis treatment with acid or enzyme). Hydrolyzate] was added or none was added, the survival rate of 90% or more was maintained until the end of the culture (Fig. 5).

When protein hydrolysates (hydrolysates produced by hydrolyzing soybean or yeast extracted proteins with acids or enzymes) were added, the protein concentration during the production incubator was up to about 40 µg / ml. 10 times increased compared to the case without addition, about 2.5 to 3 times increased compared to the case of adding sodium butyrate (Fig. 6).

As described above, the method for culturing animal cells producing the recombinant protein of the present invention is produced by hydrolyzing a protein hydrolyzate (soybean or yeast extract protein with acid or enzyme) in a serum-free medium. Hydrolyzate] is added to the culture to alleviate the cell death, while increasing the secretion rate of the protein at least five times more effective.

Claims (5)

As a method of culturing animal cells producing a recombinant protein, Preparing a hydrolyzate produced by hydrolyzing the soybean or yeast extract protein with an acid or an enzyme; Culturing the cells in serum-free medium while maintaining an oxygen concentration of at least 50% and a pH of 7.2; And Culturing the cells after adding the hydrolysis product in an amount of 3 g / L to 20 g / L when the cell concentration reaches 1.5 × 10 ⁶ cell lines / ml. The method of claim 1, Further culturing the cells in serum-free medium containing 3 g / L to 20 g / L of the hydrolyzate produced by hydrolyzing the soybean or yeast extract protein with acid or enzyme. Method comprising a. The method according to claim 1 or 2, The recombinant protein is selected from the group consisting of interferon beta (inteferon-β), erythropoietin (EPO) and coagulation factor eight. delete delete

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