KR100251521B1 - Cell culture method to increase secretion rate of recombinant protein - Google Patents

Abstract

PURPOSE: Provided is a method for culturing cell to enhance the secretion rate of a recombinant protein. The method can inhibit the repression of activity of a recombinant protein by death or crush of a cell and increase the secretion rate of a protein more than four times. CONSTITUTION: A method for culturing cell to enhance the secretion rate of a recombinant protein is composed of the following steps of: (a) growing a cell sufficiently using a medium including serum for cell growth; and (b) increasing the secretion rate of a recombinant protein using serum free medium including more than one reductant selected form cysteine, glutathione, reduced form, 2-mercaptoethanol, cysteamine or mixture thereof for producing a recombinant protein.

Description

Cell culture method to increase secretion rate of recombinant protein

The present invention relates to a cell culture method for increasing the secretion rate of recombinant protein in the production of recombinant protein through animal cell culture.

More specifically, cells are sufficiently grown using a medium containing serum as a medium for cell growth, and then cysteine, glutathione (reduced form) and mercaptoethanol as a medium for producing recombinant protein. The present invention relates to a cell culture method for increasing the secretion rate of recombinant protein per unit cell by using a serum-free medium containing at least one reducing agent such as 2-mercaptoethanol or cysteamine. By culturing the cells, the secretion rate of the protein can be increased by more than four times while preventing the degradation of the recombinant protein activity due to cell death or disruption.

Recombinant proteins used in medicine are mostly glycoproteins, and carbohydrate chains linked to specific sites of amino acids constituting the recombinant protein play an important role in protein activity and stability (Fukuda, MN et. al ., Blood , 73, 84-89 (1989); Varki, A. Glycobiology , 3, 97-130, 1993). In animal cells, the recombinant protein is secreted extracellularly, so that the exact glycosylation is performed to facilitate the purification process. Therefore, a complex eukaryotic cell such as an animal cell is used to obtain a recombinant protein having high activity and stability by fully glycosylation. It is commonly used as a host cell.

However, when using eukaryotic cells such as animal cells as host cells, there is a disadvantage in that the expression amount of recombinant protein is less than when using recombinant E. coli or recombinant yeast as host cells.

To overcome this drawback, we used selection marker genes such as Dihydrofolate Reductase (dhfr), Glutamine Synthetase (GS), and Adenine Deaminase. Gene amplification methods are used (Martin JP and Mark AS, BIO / TECHNOLOGY , 9, 64-68 (1994); Brown ME et. Al ., Cytotechology, 9, 231-236 (1992)). Gene amplification significantly increases the copy number in the cell, increasing the level of expression of the recombinant protein. However, in order for the recombinant protein to be secreted outside the cell, the protein must be accurately folded and then subjected to posttranslational modification.

Therefore, in order to obtain a large amount of recombinant protein, not only the recombinant protein must be expressed in a large amount in the cell but also must be able to secrete the expressed recombinant protein in a large amount outside the cell, that is, the culture medium.

Sodium butyrate has been used by many researchers to date to increase the expression and secretion of recombinant proteins in animal cell culture. However, the addition of sodium butylate in cell culture can increase the expression and secretion of recombinant protein, but the addition of sodium butylate stops cell growth and induces apoptosis (Palermo). DP et. Al ., J. of Biotechnology , 19, 35-48 (1991); Dorner AJ et al. , J. of Biol. Chem. , 264, 20602-20607 (1989); Chotigeat W., et. al. , Cytotechnology, 15, 217-221 (1994); Parker, MI, et. al., J. of Biol. Chem ., 261, 2786-2790 (1986); Calabresse, C. Biochem. Biophys. Commun. , 195, 31-38 (1993)).

When cells are killed or lysed, various glycolytic enzymes present in the cells are released to the cell, ie, culture medium, and the sugar chains of the recombinant protein already secreted extracellularly by these enzymes are degraded to decompose the activity of the recombinant protein. There is a side effect that is greatly reduced (Gramer, MJ et. Al. , Biotechnol. Prog. , 9, 366-373 (1993)).

Accordingly, the present inventors have continuously tried to develop a culture method capable of secreting a large amount of recombinant protein into a culture medium while suppressing cell death as much as possible, and thus, using a recombinant protein production medium having a reducing agent added to a serum-free medium, The present invention has been completed by discovering that the secretion rate of recombinant proteins can be greatly increased without cell death or disruption.

It is an object of the present invention to provide a cell culture method for increasing the secretion rate of recombinant protein in the production of recombinant protein through animal cell culture.

1 is a graph showing the secretion characteristics of EPO by CHO cells in LSF (-) medium and medium containing 3 mM reduced glutathione added to LSF (-),

2 is a graph showing the secretion characteristics of EPO by CHO cells in LSF (-) medium and medium in which one or more reducing agents are added to LSF (-) medium.

3 is a result of measuring the molecular weight of EPO secreted in the medium to which the reducing agent was added by Western blotting method,

Lane 1: a standard sample of EPO;

Lane 2: standard molecular weight marker (BIO-RAD);

Lane 3: the culture solution after 24 hours of incubation in LSF (-) medium;

Lane 4: culture medium 24 hours after culturing by adding 3 mM of reduced glutathione to the LSF (-) medium;

Lane 5: culture medium 24 hours after culturing by adding 1 mM of mercaptoethanol to the LSF (+) medium;

FIG. 4 is a graph showing the secretion characteristics of EPO by CHO cells in the culture medium added with 5 mM of cysteamine to LSF (+) medium and LSF (+) at the time of suspension culture.

In order to achieve the above object, when a recombinant protein is produced through animal cell culture, the cells are sufficiently grown using a medium containing serum as a cell growth medium, and then a reducing agent is added to a serum-free medium as a medium for producing a recombinant protein. Provided is a cell culture method for increasing the secretion rate of recombinant proteins using a medium added more than one kind.

Hereinafter, the present invention will be described in detail.

The present invention provides a cell culture method for increasing the secretion rate of the recombinant protein in the production of recombinant protein through cell culture.

According to the present invention, the growth rate of the recombinant protein is increased by using a medium containing serum as a medium for cell growth, and then using a medium in which at least one reducing agent is added to the serum-free medium as a medium for producing a recombinant protein. Increase.

Specifically, the recombinant CHO cell line (Accession No. KCTC 0220BP), which produces a recombinant erythropoietin (abbreviated as “EPO”), is sufficiently grown in a growth medium containing serum, followed by one or two reducing agents. The rate of secretion of EPO can be increased by culturing with added serum-free production medium.

In this case, cysteine, reduced glutathione, mercaptoethanol or cysteamine may be used as the reducing agent, and the amount of the reducing agent added to the medium is preferably 0.1 mM to 10 mM and more preferably 1 mM to 5 mM.

Any animal cell used in the method of the present invention may be any animal cell genetically engineered for foreign protein production, including CHO cells, and any recombinant protein may be used as long as it is a secreted protein. More preferably, proteins secreted with sugar chains are used.

Serum-free medium also includes LSF medium (Korean Patent Application No. 97-7188), such as CHO-S SFM II of GIBCO or EX-CELL ^TM 301 medium of JHH Bioscience. Any serum-free medium already known commercially can be used.

In addition, the method of the present invention is preferably used in combination of one or two or more of the reducing agent in the case of culturing the cells by the attachment culture method, it is preferable to use a combination of two or more of the reducing agent when incubated in the suspension culture method.

When culturing CHO cells by the method of the present invention, the secretion rate of EPO can be increased by four times or more as compared with the case where a medium without a reducing agent is used.

The present invention, lactate dehydrogenase (Lactate dehydrogenase, LDH) which is one of the enzymes released into the culture medium when the cells are killed or crushed when the reducing agent is added to the cell culture medium to determine whether the cells are killed or crushed Measure the activity in the culture medium.

In addition, the glycosylation change of the protein by measuring the molecular weight of the recombinant protein produced using the cell culture method of the present invention to determine whether the change in recombinant protein activity due to cell death or disruption when the reducing agent is added to the cell culture medium Investigate.

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

The following examples illustrate the present invention in detail and the present invention is not limited by the examples.

Example 1 Test of Secretion Characteristics of Recombinant Protein When Glutathione was Added to LSF Medium

In order to determine the change in secretion rate of recombinant protein when the reducing agent is added to the cell culture medium, reducing glutathione is added to the LSF medium (hereinafter referred to as “LSF (-)”) containing no reducing agent. Cell culture was performed.

Initial cell concentration in two T-25 flasks (FALCON) using CHO cells transformed to produce EPO using a medium containing 10% fetal bovine serum (FBS, GIBCO) in IMDM (GIBCO). Was inoculated to 2 × 10 ⁶ cells / T-25, and then incubated in 37 ° C. and 5% CO ₂ incubator for about 48 hours. When cells have reached sufficient growth and reached approximately 7.5-8 × 10 ⁶ cell counts / T-25, the medium was removed, washed once with PBS (GIBCO) buffer, and then LSF (-) was added to one T-25 flask. In another T-25 flask, medium was added with 3 mM of reduced glutathione to LSF (-), and culture was started. Cultures were taken from each flask at 1, 2, 4, 6, 11, and 24 hours after incubation, and the amount of EPO secreted by CHO cells from the supernatant after centrifugation was quantified using the enzyme immunoassay (EIA). See FIG. 1 ). Enzyme immunoassay is a sandwich method using monoclonal and polyclonal antibodies against EPO. The standard straight line is obtained from a known standard solution, and the absorbance of the sample solution is substituted into the standard straight line to determine the amount of EPO secreted in the medium. Protein quantitation.

If the medium does not contain a reducing agent, EPO is hardly secreted until 6 hours after incubation. After 6 hours, EPO secretion gradually increases, but when 3 mM of reduced glutathione is added to the medium, After 6 hours, the secretion of EPO rapidly increased and then gradually increased. After 24 hours, the amount of recombinant protein increased more than three times compared to the culture medium without reducing agent. When cultured in a medium containing tataion, it reached about 9 × 10 ⁶ cells / T-25.

In order to determine whether cell death or crushing occurs when a reducing agent is added to the cell culture medium, lactate dehydrogenase (LDH), one of enzymes released into the culture medium when cells are killed or crushed, Activity was measured using an LDH assay kit (Boehringermannheim. Cat. No. 1,644,793) and the results are shown in Table 1 below.

Lactate Dehydrogenase (LDH, unit / ml) Incubation time LSF (-) LSF (-) + Glutathione 3mM One 0.057 0.116 2 0.001 0.112 4 0.083 0.106 6 0.121 0.065 11 0.119 0.388 24 0.308 0.442

LDH activity was about 6-7 units / ml when the cells were completely crushed, whereas the activity was very low when glutathione was added to the medium. Able to know.

<Example 2> Secretion characteristics test of recombinant protein by each reducing agent when various reducing agents are added to LSF medium

In order to confirm the change of the EPO secretion rate by each reducing agent when several different kinds of reducing agents were added to the LSF (-) medium, 5 mM of each reducing agent, cysteine, reduced glutaion and mercaptoethanol, was added to the LSF (-) medium. ) Was added to the medium and cultured in the same manner as in Example 1.

Equation 1 was used to determine the secretion rate of EPO per unit cell.

dp / dt = qX

q = PP ₀ / ∫X dt

P: amount of EPO in the medium (µg / ml)

X: cell concentration (number of cells per ml)

q: EPO secretion rate per unit cell (㎍ EPO / 10 ⁶ cells hr)

According to the above formula, the EPO secretion rate per unit cell can be obtained by plotting the amount of EPO and ∫X dt measured by the enzyme immunoassay.

EPO secretion rate when the reducing agent is added divided by the EPO secretion rate in the medium containing no reducing agent as shown in Table 2 below.

Experiment medium Secretion rate of EPO per unit cell ¹⁾ LSF (-) 1.00 LSF (-) + Cysteine 5mM 4.20 LSF (-) + Glutathione 5mM 4.70 LSF (-) + mercaptoethanol 5mM 4.00 IMDM 10% FBS 3.70 ¹⁾ Normalized value of EPO secretion rate in LSF (-) medium

It can be seen that the EPO secretion rate per unit cell when the cell culture was added to the LSF (-) medium increased more than four times compared to when the cells were cultured in the LSF (-) medium containing no reducing agent. The addition of only one of the various reducing agents to the serum-free medium can be seen that the secretion of a larger amount of recombinant protein than in the medium containing serum.

<Example 3> Secretion characteristics test of recombinant protein when two reducing agents are added together in LSF (-) medium

In order to confirm the change of EPO secretion rate by reducing agent when two reducing agents were added together, LSF medium (LSF (-)) containing 0.3mM of cysteine and cystine, reducing agent couple, was added to LSF (-) medium. 5mM of cysteamine was added to the medium containing one reducing agent, hereinafter referred to as “LSF (+)”, and cell culture was performed by the same culture method as in Example 1.

Figure 2 shows one reducing agent added to the medium when there is no reducing agent [LSF (-)], when there is one reducing agent [LSF (+)], and when there are two reducing agents [LSF (+). Morphology] A graph showing the secretion characteristics of EPO by CHO cells. As soon as one or more reducing agents are present in the medium, EPO begins to secrete and then increases gradually until 10 hours after medium exchange. In particular, when two reducing agents are present, the secretion amount of EPO is higher than in the medium in which one reducing agent is present.

<Example 4> Secretion characteristics test of recombinant protein by each reducing agent when various reducing agents are added to LSF (+) medium

In order to confirm the change of EPO secretion rate by each reducing agent when several different kinds of reducing agents were added to the LSF (+) medium, 5mM of reducing agents, reduced glutathione, and cysteamine, respectively, and mercaptoethanol 1 mM was added to the LSF (+) medium to culture the cells by the same culture method as in Example 1. EPO secretion rate in each medium was determined in the same manner as in Example 2.

Table 3 below shows the change in the secretion rate of EPO when various reducing agents were added to the LSF (+) medium as the normalized value of the EPO secretion rate in the LSF (+) medium.

Experiment medium Secretion rate of EPO per unit cell ¹⁾ LSF (+) 1.00 LSF (+) + cysteine 5 mM 4.95 LSF (+) + Glutathione 5 mM 2.13 LSF (+) + mercaptoethanol 1 mM 1.64 LSF (+) + cysteamine 5 mM 7.38 IMDM 10% FBS 1.30 ¹⁾ Normalized value of EPO secretion rate in LSF (+) medium

In the case of cell culture by adding two reducing agents to the LSF (-) medium, the EPO secretion rate per unit cell was 1.6 times as much as 7 times higher than that of the LSF (+) medium containing one reducing agent. A greater amount of EPO was obtained than EPO secreted from the serum-containing medium.

Example 5 Determination of Molecular Weight of Recombinant Protein Secreted in a Medium Added with Reducing Agent

The molecular weight of EPO produced in the present invention was measured by Western blot method to determine whether changes in glycosylation of recombinant proteins occur when the cells were cultured using a medium containing a reducing agent. Glycosylation has a great effect on the bioactivity of the recombinant protein, and thus, complete glycosylation is necessary for the recombinant protein to have high bioactivity.

The culture solution was subjected to western blotting according to the method of ED Harlow & David Lane, Antibodies, A Laboratory Manual, 1988, Cold Spring Harbor Laboratory as follows.

After SDS-PAGE, the protein band separated on the gel was electrically transferred to nitrocellulose paper (BIO-RAD). This nitrocellulose paper was treated with PBS solution containing 1% bovine serum albumin (SIGMA) for 2 hours, washed three times with PBS solution for 3 minutes, and then monoclonal antibody against EPO (Genzyme cat. No AE7A5). Was treated for 2 hours in a 1% bovine serum albumin / PBS solution containing 500-fold dilution. After washing with PBS solution three times for 5 minutes, 1% bovine albumin / PBS containing 500-fold dilution of alkaline phosphatase conjugated goat affinity purified antibody to mouse IgG (Cappel Cat. No.59296) The solution was treated for 1 hour and washed three times with 5 minutes each with PBS solution. This nitrocellulose paper was added to 10 ml of alkaline phosphatase buffer (AP buffer (pH 9.5); 100 mM NaCl, 5 mM CaCl ₂ , 100 mM Tris) and the color reagent NBT / BCIP (5-bromo-4-chloro-3-indoleylphosphate-p) was used. Toluidine salt / nitro-bluetetrazolium chloride, Boehringer Mannheim) were added by adding 66 µl and 33 µl, respectively.

3 is a Western blot result. EPO is a glycoprotein and its molecular weight is approximately 36 KD when fully glycosylated. It can be seen that when one or two reducing agents are added to the medium, there is no significant change in the molecular weight of the EPO secreted by the CHO cells into the medium, and thus the molecular weight distribution of the recombinant protein does not change with the addition of the reducing agent. .

Example 6 Secretion Characteristics of Recombinant Proteins When Cysteamine, a Reducing Agent, is Added to LSF (+) Medium in Cell Culture by Suspension Culture

Suspension culture of CHO cells was performed in a 125 ml spinner flask. Cell suspensions deposited with accession number KCTC 0220BP for suspension culture were used after undergoing an adaptation process to grow well in suspension culture (hereinafter referred to as suspension culture cell lines).

First, inoculate the suspension culture cell line with IMDM (GIBCO Co., Ltd.) using a medium containing 10% fetal bovine serum in a 60 mm dish (Dish, FALCON Co., Ltd.) to give an initial cell concentration of 1.6 × 10 ⁶ cells / dish. Incubated for about 4-5 days at 37 ℃, 5% CO ₂ incubator. After confirming the cells were spread out and grown, trypsin (0.25%, GIBCO) was used to remove the cells from the dish and the initial cell concentration was 2 × 10 ⁵ / ml in two spinner flasks containing 100 ml of LSF (+) medium. Inoculation was carried out so that the culture was started at a rotational speed of 50 rpm in 37 ℃, 5% CO ₂ incubator. After 4 days of culture, when the cell concentration reached 1 × 10 ⁶ / ml, the medium of one spinner flask was discarded by discarding the culture solution, and the precipitated cells were resuspended in fresh LSF (+) medium, 5 mM of amine was added and transferred to a spinner flask and incubated.

4 shows EPO secretion in LSF (+) medium during suspension culture and the amount of EPO secretion when cysteamine is added to LSF (+) medium. In Example 3, EPO was rapidly secreted even in LSF (+) medium to which one reducing agent was added during the attachment culture, but EPO in LSF (+) medium to which one reducing agent was added in suspension culture. Secretion rarely occurred. On the other hand, when 5 mM of cysteamine was added to the LSF (+) medium, the secretion of EPO proceeded rapidly after 5 hours.

As described above, in suspension culture, two or more reducing agents may be added to greatly increase the secretion rate of EPO.

As described above, cell culture in a medium in which one or two or more reducing agents are added to a serum-free medium that does not contain a reducing agent may increase the secretion rate of the recombinant protein by four times or more. Can be mass-produced, and the activity of the recombinant protein can also be suppressed by cell death or disruption.

Claims (5)

Cells are sufficiently grown using a medium containing serum as a cell growth medium, and then cysteine, reduced glutathione, mercaptoethanor cysteamine or mixtures thereof in serum-free medium as a medium for producing recombinant proteins. Cell culture method for increasing the secretion rate of the recombinant protein using a medium to which at least one reducing agent is selected from the group comprising. The cell culture method of claim 1, wherein the amount of cysteine is 0.1-10 mM. The cell culture method of claim 1, wherein the amount of glutathione used is 0.1-10 mM. The method of claim 1, wherein the amount of mercaptoethanol is 0.1-10 mM cell culture method. The cell culture method according to claim 1, wherein the amount of cysteamine used is 0.1-10 mM.

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