KR100394474B1 - Process for the continuous mass-production of recombinant glycoprotein in animal cells - Google Patents

Abstract

The present invention relates to a method for culturing animal cells producing a recombinant glycoprotein, and specifically, the method for culturing the present invention comprises a growth step of culturing the animal cells in a serum growth medium and a serum-free production medium containing an inducer. The recombinant glycoprotein production step of culturing animal cells is characterized in that it is continuously repeated, and by using the culturing method of the present invention it is possible to mass-produce a highly active recombinant glycoprotein.

Description

Process for the continuous mass-production of recombinant glycoprotein in animal cells

The present invention relates to a method for culturing animal cells producing recombinant glycoproteins, and more particularly, to a step of culturing the animal cells in a serum growth medium, and to culturing the animal cells in a serum-free production medium containing an inducer. It relates to a method for culturing animal cells, characterized in that to continuously repeat the recombinant glycoprotein production step.

Animal cell culture is a key technology for producing high value-added biological drugs such as therapeutics and diagnostics using genetic recombination technology and cell fusion technology. It uses expensive medium and grows slower than microbial culture. Due to the high cost and lack of cell walls, attention should be paid to agitation and aeration, and great care must be taken to industrially use the process, such as fear of contamination by viruses, etc. Translational modification and the ability to create three-dimensional structures and easily fold proteins are used as important technologies in the production of some biopharmaceuticals. Examples of the production include unicellular antibody, erythropoietin (EPO), and tissue-type plasma activator (tPA), which are expected to greatly expand research on the optimal mode of operation of bioreactors seeking high productivity.

Common animal cell cultures are divided into adherent cell cultures and floating cell cultures, and industrial production of useful materials requires scale-up of 100 L, 1000 L or more from 1 L systems. Scaling up is not enough to simply scale up the size of the culture vessel and requires optimization of various factors, such as pH, nutrients, and oxygen, that provide a suitable growth environment.

In the case of adherent animal cells, most of them have traditionally carried out mass cultivation by roller culture vessels. The roller cultivation method is a common and easy method, and the process steps such as medium injection are clear, so it is easy to distinguish between lots. It is possible to prevent the entire culture from being contaminated at once, and has the advantage of being able to produce and separate at the same time, and also the scale-up efforts for mass production since the small quantity production and mass production are determined by the number of roller culture vessels. This is not necessary. However, most animal cell productivity is closely related to the number of viable cells. Roller culture vessels have a limited surface area and cannot provide a suitable growth environment for permanently proliferating production cells. Difficult to control the supply of nutrients, oxygen, etc. has a problem that the cells die quickly. In particular, EPO, a glycoprotein, is known to exhibit high activity in vivo as the sialic acid or N-acetylneuraminic acid (glyc-terminated sialic acid or N-acetylneuraminic acid) increases its in vivo activity. Glycosidase, an enzyme that breaks down intracellular sugar chains, and especially sialidase that breaks down sialic acid sugar chains, when released into cells, reduces the sialic acid content of EPO that is produced in culture. It has the problem of lowering the quality of EPO in the culture stage. Therefore, roller culture has a disadvantage in that the production must be carried out in a batch culture with a short production period, and a large number of roller culture vessels and labor are required for mass production of a desired amount, which has a disadvantage in economic efficiency.

In the present invention, a method of continuously producing recombinant glycoproteins in a roller culture vessel was established as a method to compensate for such disadvantages. In other words, by continuously repeating the recovery period of the production cell line and the production of recombinant glycoproteins during the culturing process, a continuous roller culture process that can continuously produce high-quality recombinant glycoproteins without damaging or dying cells was invented. This is a process to collect the supernatant produced continuously for a long time in a single inoculation can periodically control the number of cells attached to the roller container can provide a suitable growth environment in the roller container culture.

In addition, in the present invention, in order to maximize expression of recombinant CHO cell lines and BHK cell lines, the expression medium is expressed by adding n-butyric acid or methotrexate (hereinafter, abbreviated as 'MTX') as an expression inducing agent. The present invention has been completed by maximizing the amount.

An object of the present invention is to increase the productivity of recombinant proteins by utilizing and improving existing methods for mass production of recombinant glycoproteins using roller culture in serum-free medium.

1 shows the results of EPO production before and after induction of n-butyric acid in the T-flask of the recombinant CHO cell line, DCHE715 and recombinant BHK cell line, DBHE318, used in the present invention.

2 is an overall process diagram of the continuous mass production method,

Figure 3 shows the results of CHO-EPO, BHK-EPO production of serum-free continuous roller culture,

Figure 4 is the production of CHO-EPO, BHK-EPO production of serum-free batch roller culture,

Figure 5 shows the survival rate of cells in the production of CHO-EPO, BHK-EPO in serum-free continuous roller culture,

Figure 6 shows the survival rate of cells in the production of CHO-EPO, BHK-EPO in serum-free batch roller culture.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for culturing animal cells producing recombinant glycoproteins. The culture method is characterized in that the production of recombinant glycoproteins is increased by repeating the growth of animal cells and the production of recombinant glycoproteins. More specifically, the characteristics of the culture method of the present invention

1) a growth step of culturing the animal cells in a serum growth medium,

2) characterized in that the recombinant glycoprotein production step of culturing animal cells in a serum-free production medium containing an inducer is continuously repeated.

In the animal cell culture method of the present invention, the glycoprotein production step and growth step are repeated at least once. The culture period of each stage can be variously selected depending on the animal cell used, the specific composition of the culture medium, and other culture conditions.However, when the cell concentration of the culture medium decreases below 70-80% at the beginning of the production stage, Preferably, for example, the glycoprotein production step for 2 to 4 days, the growth step for 2 to 3 days can be repeated to culture the animal cells.

In the animal cell culture method of the present invention, the animal cells to be cultured are cells used to produce recombinant glycoproteins, wherein the recombinant glycoproteins are all glycoproteins that can be used in the therapeutic and diagnostic medicines of humans, in particular, erythro Poietin (EPO), or Tissue-type Plasminogen Activator (tPA), Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), Interleukin-12 (Interleukin- 12, IL-12), human chorionic gonadotropin (hCG), thrombopoietin (TPO), anticoagulant factor-8 (antihemophilic factor, Factor VII), etc. may be preferably used. have.

The animal cells include all animal cells that can be used for expression of glycoproteins. Animal cells used in the animal cell culture method of the present invention are particularly referred to as Chinese Hamster Ovary (hereinafter referred to as 'CHO'). Cells and Baby Hamster Kidney (hereinafter abbreviated as 'BHK') cells are preferred. CHO and BHK cell lines used in the present invention are nucleosides with cell lines deficient in dihydrofolate reductase (hereinafter abbreviated as 'DHFR'), a major enzyme involved in nucleoside synthesis. MEM-α can not survive without a medium. By introducing a plasmid linking the gene of the target protein to the gene of the DHFR into the animal cell, only the cell in which the gene was introduced in the MEM-α medium can survive, so that only the transformed cell is naturally selected after a certain period of time. In particular, since MTX, a folate analog, inhibits nucleoside synthesis, the addition of MTX to the medium induces amplification of DHFR and target genes introduced from outside to survive. Production of recombinant human EPO glycoprotein hormone can also be amplified.

In the animal cell culture method of the present invention, the serum-free production medium of the production step may be used all known serum-free medium for eukaryotic cell culture, and includes various known inducers. Here, the inducer is a substance capable of controlling the expression efficiency of the gene to be expressed in the animal cell, a substance capable of activating a foreign promoter regulating the gene, and amplifying a copy number in the genome of the gene. Inclusively refer to the material that can be made. The specific composition of the inducer included in the serum-free production medium in the present invention is determined according to the expression vector introduced into the eukaryotic cell, specific examples of the inducer are G418, Hygromycin-B, Aminopterin, Mycophenolic acid, 9-β-D-Xylofuranosyl adenine, n-butanoic acid, methotrexate (MTX). Preferred inducers in the animal cell culture method of the present invention include n-butanoic acid or MTX, and the concentration is preferably 3 to 24 mM n-butanoic acid or 0.1 to 10 μM MTX, and particularly 6 to 12 mM n-butanoic acid is used. desirable.

DCHE715 (Korean Patent Application No. 98-32997, Accession No. KCLRF-BP-00018), a recombinant CHO cell line used in the present invention, and DBHE318 (Korean Patent Application No. 99-37463, Accession No. KCLRF-BP-00024), a BHK cell line The gene can be amplified by n-butanoic acid as well as MTX. n-butanoic acid can regulate the expression of several specific genes in various types of cells (Kruh et al., J. Mol. Cell Biochem., 42: 65, 1985). Increased plasmid DNA expression (Gorman et al., Nucleic acid res., 11: 7631, 1983) or the expression of plasmid DNA containing the SV40 early promoter-CAT (chloramphenicol acetyl transferase) transcription structure. (Ken et al., Cell structure and function, 14: 579, 1989). In addition, when n-butanoic acid was treated to several cells, the cell growth was inhibited, the shape of the cell was changed, and when n-butanoic acid was removed, it returned to its previous state after a recovery period of 20 to 25 hours (Kruh). et al., J. Mol. Cell Biochem., 42:65, 1985). The characteristics of n-butanoic acid showed inhibition of cell growth and transient expression of recombinant protein of DCHE715 and DBHE318 used in the present invention and increased up to 20-fold in T-flask culture (see FIG. 1 ).

In the animal cell culture method of the present invention, the culture of the animal cells is preferably carried out by adhesive roller culture, because the roller culture method is a general and easy method, and the process steps such as medium injection are clear, so that between lots It is easy to distinguish, prevents the whole culture from being contaminated at once, has the advantage of being able to produce and separate at the same time, and also because the small quantity production and mass production are determined by the number of roller culture vessels for mass production This is because there is no need to scale up.

Figure 2 shows schematically an example of the animal cell culture method provided by the present invention. According to the culture method shown in Figure 2 , the seed cell culture can be carried out as a step before producing the recombinant glycoprotein. Various well-known seed culture methods can be used in the animal cell culture method of the present invention, and the seed culture method specified in Examples or Comparative Examples of the present invention is not presented for the purpose of limiting the animal cell culture method of the present invention. .

As can be seen in Figure 2 , the culture supernatant obtained from the animal cell culture method of the present invention can be used to produce the desired recombinant glycoprotein through filtration and the like.

According to the Examples and Comparative Examples of the present invention, the production results of the production results in the batch culture method of the recombinant CHO cell line DCHE715 and BHK cell lines DBHE318 obtained by culture of serum-free roller and a continuous culture method is shown in Figures 3 and 5 respectively, same.

In the case of batch production, the production period of CHO-EPO and BHK-EPO by induction of n-butanoic acid was 6-8 days, and in case of CHO-EPO production, 7,113 international per ml of culture supernatant on day 2 after induction of n-butanoic acid The unit (international unit, hereinafter abbreviated as 'IU') showed the highest production of CHO-EPO during the incubation period. In addition, the production of BHK-EPO also showed the highest production of BHK-EPO during the incubation period at 8,689 IU per ml of culture supernatant 2 days after n-butanoic acid induction. The average daily production of CHO-EPO was 3,036 IU per ml of culture supernatant, and finally the total CHO-EPO produced per roller culture vessel was more than 4,857,600 IU. The average daily production of BHK-EPO was culture supernatant 1 It was 3,794 IU per ml, and finally the total amount of BHK-EPO produced per roller culture vessel was 6,070,000 IU or more.

On the other hand, in the continuous culture carried out in the embodiment of the present invention, the production period of CHO-EPO and BHK-EPO by induction of n-butanoic acid was 20 days at 10 times, 2 days at 1st, and n during 1 ~ 10th. On day 2 after induction of butanoic acid, the highest EPO production was achieved at a maximum of 37,683 IU per ml of CHO-EPO culture supernatant and 29,563 IU per ml of BHK-EPO culture supernatant. The average daily production of CHO-EPO was 20,658 IU per ml of culture supernatant, and the average daily production of BHK-EPO was 17,321 IU per ml of culture supernatant. Finally, the total amount of CHO-EPO produced per roller culture vessel was 82,632,000 IU or more, and the total BHK-EPO was more than 69,284,000 IU.

On the other hand, serum-free roller cultures can be equipped with 45 1750 cm ² roller bottles per roller incubator, and each day from day 1 to day 2 after induction agent treatment per 1750 cm ² roller bottle. 200 ml of the culture was recovered 10 times. Therefore, in the present invention, it is possible to produce high CHO-EPO of about 3,718,440,000 IU or more per 1750 cm ² roller bottle, and to produce high EPO of 3,117,780,000 IU or more for BHK-EPO. Continuous roller culture was able to produce high yields of about 17 times for CHO-EPO and about 11.5 times for BHK-EPO than conventional batch roller culture.

In the case of batch roller cultivation, the survival rate of cells dropped rapidly to 50% or less after 3-4 days of production, and it is difficult to produce high-quality EPO. It has been shown that the quality of EPO can be produced (see Figs. 4 and 6 ).

According to the results, as in the animal cell culture method of the present invention, the growth step of culturing the animal cells in the serum-free growth medium and the recombinant glycoprotein production step of culturing the animal cells in a serum-free production medium containing an inducer If repeated, it is possible to produce a recombinant glycoprotein having a higher yield in higher yield than when producing the desired glycoprotein by simply culturing the same animal cells in a batch.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Serum-free Continuous Roller Culture and Production

(1-1) spawn culture

Recombinant CHO cell line DCHE715 (Korean Patent Application No. 98-32997, Accession No. KCLRF-BP-00018) stored in a liquid nitrogen tank or Recombinant BHK cell line DBHE318 (Korean Patent Application No. 99-37463, Accession No. KCLRF-BP- 00024) were taken out of 1 vial (1 × 10 ⁷ cells / ml) and dissolved as quickly as possible in a 37 ° C constant-temperature water bath, followed by the seed culture medium (α-MEM containing 10% serum and 0.5 μM MTX, cath. 10 L) was washed once and centrifuged to inoculate one 175 cm2 T-flask (cat # 431080, Corning, USA) containing 50 ml of the seed culture medium. Incubate for 2-4 days in a CO ₂ incubator (37 ° C., 5% CO ₂ ) to sub-confluency, then treat 0.25% Trypsin-EDTA solution in a 175 cm 2 T-flask. The cells were recovered. Cells recovered from one T-flask were inoculated in three new 175 cm 2 T-flasks containing 50 ml of fresh seed culture medium.

(1-2) Main culture

One 1750 cm 2 roller culture vessel containing 200 ml of fresh growth medium (10% serum containing DMEM / F12, JRH, cat. # 56498-10L) was recovered from two 175 cm 2 T-flasks (US Corning). Inc., cat. # 25150-1750) and incubated in a roller incubator (Bellco, USA, model # 7630-82509) and incubated for 2 to 3 days at 37 ℃, 0.5rpm until sub-confluency Cells were then harvested by treatment with 0.25% trypsin-EDTA solution. Cells recovered from one 1750㎠ roller culture vessel were divided and inoculated into three new 1750㎠ roller culture vessels containing 200 ml of fresh growth medium, mounted on the roller incubator, and saturated at 37 ° C. and 0.5 rpm. Incubated for 2-3 days.

(1-3) Production of Glycoproteins

1) Primary culture

In the main culture, when the cells grew to near-saturated state (3.5 × 10 ⁸ cells or more), serum-containing medium was recovered and 200 ml of serum-free medium containing an inducer was added to induce CHO-EPO or BHK-EPO production. . At this time, 3 ~ 24mM n-butanoic acid or 0.1 ~ 10μM MTX was used as an inducer, and EX-cell 301 medium (Cat. # 14311-10L, USA) was used as a serum-free medium.

The culture supernatant was recovered from the roller culture vessels at 1 day (24 hour) intervals and exchanged with 200 ml of serum-free medium containing fresh n-butanoic acid, until the recombinant CHO cell line and the recombinant BHK cell line survived more than 80%. That is, it was cultured for 2 days. At this time, the rpm of the roller incubator was 1.5. After sterile filtration of the culture supernatant of CHO-EPO or BHK-EPO recovered at daily intervals, the titer of human EPO was measured by the method of Experimental Example 1 and stored at 4 ° C.

2) Continuous Culture

After 2 days of primary production, the medium is exchanged again with a growth medium (DMEM / F12 containing 10% serum, cat. # 56498-10L, 10% serum) in a 1750 cm 2 roller culture vessel. Incubate by incubating for 2 to 3 days at 0.5 rpm in a roller incubator at 37 ° C until the number of cells in the 1750 cm2 roller culture vessel reaches 3.5 × 10 ⁸ or more again, and then contains n-butanoic acid as the conditions of the primary production Serum-free medium was induced for 2 days, and the culture supernatant was recovered from the roller culture vessel at daily intervals. In this way, the culture supernatant is recovered for a total of 20 days or more in a growth culture for 2 to 3 days and a production culture for 2 days for 10 days. After sterile filtration of the culture supernatant of CHO-EPO recovered at daily intervals, the titer of human EPO was measured by the method of Experimental Example 1 and stored at 4 ° C.

<Comparative Example 1> spawn culture and serum-free batch roller culture

As in Example 1, after the spawn culture and the main culture, the cells were grown to a semi-saturated state (3.5 X 10 ⁸ cells or more) in the 1750 cm 2 roller culture vessel in the main culture, and the medium containing serum was recovered and the inducing agent. Each serum-free medium containing 200ml was added to induce CHO-EPO or BHK-EPO production. In this case, 3 to 24 mM n-butanoic acid or 0.1 to 10 μM MTX was used as an inducer. As a serum-free medium, EX-cell 301 medium (US JRH Co., cat. # 14311-10L) was used, and the rpm of the roller incubator was 1.5.

The culture supernatant was recovered from the roller culture vessel at 1 day intervals (24 hours) and exchanged for 200 ml of serum-free medium containing fresh n-butanoic acid for 6-8 days until the recombinant CHO cell line and recombinant BHK cell line survived. It was. After sterile filtration of the culture supernatant of CHO-EPO or BHK-EPO recovered at daily intervals, the titer of human EPO was measured by the method of Experimental Example 1 and stored at 4 ° C.

Experimental Example 1 Measurement of EPO Titer by Enzyme Immunoassay

100 μl of each diluent was added to each well of the antibody adsorption plate in the enzyme immunoassay kit (R & D Systems, Quantikine ^™ IVD ^™ ELISA, cat. # DEP00), and the standard solution, international standard solution and sample solution 100 After adding well, the solution was reacted at room temperature for 1 hour. At this time, the international standard solution was a formulation buffer solution containing human EPO international standard (86 IU / ampoule, NIBSC UK, cat. # 87-684) added human serum albumin [3% D-mannitol, 34.2mM sodium chloride, 12.5mM anhydrous] Sodium dihydrogen phosphate (pH7.0), and serum serum albumin (1.0 mg / ml)] were dissolved in 1 ml, and 10 µl per vial was dispensed and stored at -70 ° C. 1 vial (10 µl) at -70 ° C when used. Take out, melt and dilute with diluent to use.

The remaining reaction solution of each well was removed, washed 250 µl three times with a washing solution, and then 200 µl each of the enzyme antibody conjugate solution was added to each well, followed by reaction at room temperature for 1 hour. After the reaction, the reaction solution was removed, washed 200 μl three times with a washing solution, and all the remaining solution was removed. Then, 200 μl each of the prepared substrate-coloring solution was added, followed by reaction at room temperature for 20 minutes. After the reaction was completed, the reaction solution was stopped by adding 100 µl of the reaction solution to each well, the mixture was mixed well, and the absorbance was measured at 450 nm.

A standard curve was prepared using the horizontal axis and the absorbance (A _{450 nm} ) as the vertical axis, and the human EPO titer (mIU / ml) was calculated from the absorbances of the international standard and the sample solution. The average value of the three dilution titers obtained by multiplying each dilution ratio is calculated, and the measured values of the international standard and the sample are obtained. The measured values of the international standard are adjusted to 86 IU / ml, and the correction ratio is calculated. The final titer of the sample solution was calculated by multiplying the maintenance.

In the same manner as described above, the production results of CHO-EPO of recombinant CHO cell line (DCHE715) and BHK-EPO of recombinant BHK cell line (DBHE318) were measured using the serum-free roller culture of Example 1 and Comparative Example 1. The results are shown in Tables 1 and 2 below.

Production of Glycoprotein by Continuous Serum-free Roller Culture (Example 1) After n-butanoic acid induction Production of CHO-EPO of Recombinant Cell Line DCHE715 (IU / ml) BHK-EPO Production of Recombinant Cell Line DBHE318 (IU / ml) Degree Culture Primary Day 1 4,904 3,734 Day 2 9,730 6,468 Secondary Day 1 6,798 3,688 Day 2 10,737 7,025 3rd Day 1 8,800 5,023 Day 2 15,829 9,865 4th Day 1 16,091 7,065 Day 2 24,344 15,234 5th Day 1 21,386 14,076 Day 2 25,083 24,680 6th Day 1 20,257 18,907 Day 2 29,878 24,465 7th Day 1 24,232 25,465 Day 2 32,171 28,956 8th Day 1 19,022 22,569 Day 2 27,578 27,578 9th Day 1 30,005 26,849 Day 2 37,683 29,563 10th Day 1 19,461 19,461 Day 2 29,166 25,678

Production of Glycoproteins by Batch-Free Serum Roller Culture (Comparative Example 1) Incubation date after n-butanoic acid induction Recombinant cell line DCHE715 CHO-EPO production (IU / ml) Recombinant Cell Line DBHE318 BHK-EPO Production (IU / ml) Day 1 2,397 2,820 Day 2 7,113 8,689 3rd day 6,272 6,119 Day 4 3,512 4,055 Day 5 2,971 2,900 6th day 1,263 2,040 7th day 763 1,856 8th day 611 1,788

As can be seen in Table 1 and Table 2, 4,857,600 IU of CHO-EPO and 6,070,000 IU of BHK-EPO were produced for each roller culture vessel for batch serum-free roller culture, and roller culture for continuous serum-free roller culture. Each container produced 82,632,000 IU CHO-EPO and 69,284,000 IU BHK-EPO. In other words, CHO-EPO has improved productivity by about 17 times and BHK-EPO about 11.5 times. This result shows that continuous serum-free roller culture is an effective method for producing recombinant glycoprotein in large quantities. . (See Figures 3 and 4)

Experimental Example 2 Cultured Animal Cell Survival Test

Cell number measurement was determined by the dye exclusion method (FRESHNEY RI, Culture of animal cells: A Manual of Basic Technique (3rd Edition), p48 Wiley-Liss, New York, 1994). The surface of the 1750 cm 2 roller container was washed with 100-200 ml of Dulbecco's phosphate buffered saline (DPBS) and then treated with 50 ml of 0.25% Trypsin-EDTA. After 1-2 minutes, the cells separated into single cells were centrifuged at 1000 rpm for 5 minutes, and then the supernatant was removed and resuspended in DPBS. After dilution and staining 1: 1 using 0.4% Trypan blue (Trypan blue), hemocytometa (Hemocytometer) was measured by measuring the total number of cells and the number of live cells not stained to calculate the survival rate. The cell survival rate formula is as follows.

% Survival = (live cells / total cells) X 100

By virtue of Example 1 and Comparative Example 1, the survival rate of the recombinant cells according to the culture time was examined through continuous and batch-free serum-free culture, and the results are shown in Tables 3 and 4 below (see FIGS. 5 and 6). ).

Cell Survival Rate According to Incubation Time in Continuous Serum Roller Culture (Example 1) Incubation time (days) Survival rate of recombinant cell line DCHE715 (%) Survival rate of recombinant cell line DBHE318 (%) Primary 81.2 78.2 Secondary 79.7 79.4 3rd 82.6 80.3 4th 90 83.2 5th 81.4 84.5 6th 83.7 82.1 7th 79.2 78.5 8th 77.3 79 9th 78 78.2 10th 76.1 72

Cell Survival Rate According to Incubation Time in Batch-Free Serum Roller Culture (Comparative Example 1) Incubation time (days) Survival rate of recombinant cell line DCHE715 (%) Survival rate of recombinant cell line DBHE318 (%) One 100 100 2 81.2 78.2 3 63.3 59.7 4 30 44.1 5 11 31.6 6 7.5 23.5 7 - 15.2 8 - 8.1

As can be seen from Table 3 and Table 4, in the case of batch-free serum roller culture, the survival rate of the cells rapidly dropped to 50% or less after 3-4 days after production started, and it was confirmed that the production of high-quality EPO was difficult. On the other hand, in the case of continuous serum-free roller culture of the present invention, the survival rate of the cells for 20 days or more is maintained at 75% or more, it can be seen that effective in mass production of glycoproteins.

By using the method for culturing the animal cells producing the recombinant glycoprotein of the present invention, not only the titer of the recombinant glycoprotein is higher than that of the conventional batch culture method or the continuous culture method, and thus the cell survival rate is maintained at a high level for a long time. It can be usefully used for mass production of glycoproteins to be produced under high survival rate of cultured cells.

Claims (8)

In the culture method of animal cells producing recombinant glycoproteins using roller culture, i) culturing the animal cells in a serum growth medium (growth step); And ii) A method of culturing animal cells producing recombinant glycoproteins by continuous roller culture in which the animal cells are cultured in a serum-free production medium containing an inducing agent (production of recombinant glycoproteins). The method of claim 1, wherein the recombinant glycoprotein is erythropoietin (EPO), tissue plasminogen activator (tPA), follicle stimulating hormone (FSH), luteinizing hormone (LH), interleukin-12 (IL-12 ), Human chorionic gonadotropin (hCG), thrombopoietin (TPO) or blood coagulation factor-8 (Factor VII). The method of claim 1, wherein the animal cells are CHO cells or BHK cells. The method of claim 1, wherein the inducer is n-butanoic acid or methotrexate (MTX). The method of claim 1, wherein the production step of the recombinant glycoprotein is terminated and enters a growth phase when the cell concentration decreases to about 70-80% or less of the initial cell concentration. The method of claim 1, wherein the recombinant glycoprotein production step is performed for 2 to 4 days, and the growth step is performed for 2 to 3 days. The culture method according to claim 1, wherein the culturing is performed by adhesive roller culture. The method of claim 7, wherein the roller culture is performed at a condition of 1 to 2 rpm in the production stage of the recombinant glycoprotein and 0.2 to 1 rpm in the growth stage.