KR20230054218A - Method for culturing a cell expressing a protein - Google Patents

Abstract

The present invention relates to a method for culturing a cell line expressing a target protein, which is a polypeptide represented by the amino acid sequence shown in SEQ ID NO: 1 or a polypeptide having at least 75% sequence homology thereto. More specifically, the present invention provides a culture method comprises a step of culturing the cell line expressing the protein, wherein the step of culturing the cell line includes adjustment of the pH of a culture medium. In addition, the culture method of the target protein according to the present invention can be used to mass-produce the target protein while reducing the production cost of the target protein.

Description

Culturing method of cell expressing protein {METHOD FOR CULTURING A CELL EXPRESSING A PROTEIN}

The present invention is a method for culturing a cell line expressing a protein capable of mass-producing a target protein while reducing production costs, and in particular, prevents and/or infects SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus-2) infection. It relates to a method for culturing a cell line expressing an antigenic protein of a vaccine used for the purpose of alleviating symptoms expressed at the time of application.

The SARS-CoV-2 virus, which has been reported as a pandemic infection since December 2019, is a virus belonging to the Coronaviridae family, Betacoronavirus genus Sarbecovirus subgenus. It is known that the Receptor Binding Domain (RBD) of B. binds to the ACE2 receptor protein on the surface of human cells to cause infection. Spike glycoprotein monomers are separated into S1 subunits and S2 subunits by host cell proteases.

It is known that the main transmission route of the SARS-CoV-2 virus infection is close contact with droplets of an infected person. However, it is known that the virus may be transmitted by direct contact with an infected person or by touching a medium such as an item contaminated by droplets of an infected person and then touching eyes, nose, mouth, etc. without washing hands.

Meanwhile, as the social and economic damage caused by the SARS-CoV-2 virus infection intensified in 2020, emergency approval and approval for the use of vaccines that have the effect of alleviating the symptoms of SARS-CoV-2 virus infection in various countries such as the United States and Europe Together, vaccinations have been initiated in many countries around the world. However, even if the vaccine is vaccinated, SARS-CoV-2 virus infection in the vaccinated person cannot be prevented from the source.

Moreover, SARS-CoV-2 virus mutations such as alpha mutation, beta mutation, gamma mutation, epsilon mutation, delta mutation, kappa mutation, and eta mutation continue to be reported, and these mutated SARS-CoV-2 viruses in vaccinated persons Infections continue to occur. Therefore, for the purpose of maintaining an effective antibody level in the body, additional vaccination for vaccinated persons is recommended. Furthermore, governments of each country are expanding the target of SARS-CoV-2 vaccination to adolescents in consideration of the social and economic impact of SARS-CoV-2 infection. Demand for the production of antigenic proteins that can be used is rapidly increasing. A method for mass-producing a target protein in plants is already known (Patent Publication No. 10-2021-0117808), but research on a method for mass-producing a target protein in animal cells is still lacking. Therefore, it is required to develop a production method capable of mass-producing vaccine antigen proteins in animal cells in a short period of time.

(Patent Document 1) Patent Publication No. 10-2021-0117808

While conducting research on a method for increasing the production efficiency of vaccine antigen proteins, the present inventors, in the process of culturing a cell line expressing a target protein in a bioreactor, adjusted the pH of the culture medium to a certain value or less to improve culture stability and cell growth. It is possible to reduce the production cost by increasing the protein expression efficiency of the culture medium, and adjusting the injection speed of the gas introduced into the culture medium makes it possible to scale up the culture medium volume and suppress the formation of bubbles in the culture medium. The present invention was completed by finding that the productivity of proteins can be improved.

According to one aspect of the technology disclosed by the present application, a method for culturing a cell line expressing a target protein includes culturing the cell line expressing the protein, wherein the culturing the cell line adjusts the pH of the culture medium It provides a culture method comprising doing.

In addition, according to one aspect of the technology disclosed by the present application, the step of culturing the cell line may enable scaling-up of the volume of the culture medium and inhibition of foam formation in the culture medium by adjusting the injection rate of gas introduced into the culture medium.

The method for culturing a cell line expressing a protein according to the present invention can be used to mass-produce the protein while reducing the production cost of the protein.

Figure 1 shows the structure of a vector containing a nucleotide sequence encoding the protein of SEQ ID NO: 1.
FIG. 2 shows changes in dissolved oxygen (DO), pH, oxygen flow rate, and carbon dioxide flow rate in the culture solution when the upper pH value of the culture solution in the bioreactor was set to pH 7.25.
3 shows changes in DO, pH, oxygen flow rate, and carbon dioxide flow rate in the culture medium when the upper pH value of the culture medium in the bioreactor was lowered to pH 7.1, and the trend of the accumulated sodium bicarbonate consumption during the cultivation period.
Figure 4 shows the change in the amount of lactic acid produced in the culture medium when the upper pH value of the culture medium in the bioreactor is changed to 6.8, 6.99, 7.0 or 7.2, respectively.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention is a method for culturing a cell line expressing a protein of interest, comprising culturing the cell line expressing the protein, wherein culturing the cell line comprises adjusting the pH of the culture medium. provides a way

The "target protein" is any kind of exogenous product protein that can be expressed by cells. Typically, insulin, cytokine (interleukin, tumor necrosis factor, interferon, colony stimulating factor, chemokine, etc.), erythropoietin, antigen, antibody, antibody fragment, structural protein, regulatory protein, transcription factor, toxin protein, hormone, hormone Analogs, enzymes, enzyme inhibitors, transport proteins, receptors (e.g., tyrosine kinase receptors, etc.), fragments of receptors, biological defense inducers, storage proteins, movement proteins, exploitive proteins, reporter proteins, growth factors, etc., and such a target protein includes a "cloning site", which is a nucleic acid sequence into which a restriction enzyme recognition or cleavage site is introduced to allow insertion of the gene encoding the target protein into a vector for expression in a cell line. can do.

In the present invention, the target protein is one of the polypeptides described in claim 1 of International Patent Publication No. 2021-163438, International Patent Publication No. 2019-169120, US Patent Publication No. 9630994, International Patent Publication No. 2021-163481, It may be one of the polypeptides disclosed in the specification of International Patent Publication No. 2021-163438 or International Application No. PCT/US2021/037341. Preferably, the target protein may be a polypeptide having an amino acid sequence represented by SEQ ID NO: 7, 29, 30, 31, or 39 disclosed in International Patent Publication No. 2021-163438.

In one embodiment of the present invention, the target protein is a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 1, or 70% or more, 75% or more, 80% or more, 85% or more of the amino acid sequence represented by SEQ ID NO: 1 At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence homology. there is. The target protein is encoded by a gene consisting of the nucleotide sequence represented by SEQ ID NO: 2, or 70% or more, 75% or more, 80% or more, 85% or more, 90% or more of the nucleotide sequence represented by SEQ ID NO: 2, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or 99% or more sequence homology can be encoded by a base sequence.

The cell line producing the target protein is a polypeptide represented by the amino acid sequence represented by SEQ ID NO: 1 or more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 91%, more than 92% , It may be a cell line having an expression vector containing a gene encoding a polypeptide having at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence homology. . In one embodiment of the present invention, the expression vector may be a plasmid vector, but the polypeptide represented by the amino acid sequence represented by SEQ ID NO: 1 or 70% or more, 75% or more, 80% or more, 85% or more , which encodes a polypeptide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence homology Any vector suitable for transfecting a gene may be used without limitation.

Meanwhile, in the present invention, an expression vector including a gene encoding a protein represented by the amino acid sequence represented by SEQ ID NO: 1 may have a DNA sequence represented by SEQ ID NO: 3.

As used herein, the term "vector" refers to a nucleic acid means comprising a nucleotide sequence that can be introduced into a host cell and recombined and inserted into the genome of the host cell, or can be spontaneously replicated as an episome. Suitable expression vectors include expression control elements such as promoters, initiation codons, stop codons, polyadenylation signals and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and can be prepared in various ways depending on the purpose. . When a genetic construct encoding a target protein is administered, the initiation codon and stop codon must be functional in the subject and must be in frame with the coding sequence.

The polynucleotide or vector according to one embodiment of the present invention is an independent molecule outside the genome, specifically a molecule capable of replicating, and may exist in a genetically modified host cell or non-human host organism, or may be present in a host cell or non-human host cell. -Can be stably integrated into the genome of human host organisms.

A host cell of a cell line expressing a target protein according to one embodiment of the present invention is a eukaryotic cell. The eukaryotic cells include fungus cells, plant cells or animal cells. Examples of the fungal cell may be yeast, specifically Saccharomyces genus ( Saccharomyces sp ) yeast, more specifically Saccharomyces cerevisiae ( S. cerevisiae ). In addition, examples of animal cells include insect cells or mammalian cells, and specific examples of animal cells include HEK293, 293T, NSO, Chinese hamster ovary cells (CHO), MDCK, U2-OSHela, NIH3T3, MOLT-4, Jurkat, PC-12, PC-3, IMR, NT2N, Sk-n-sh, CaSki, C33A, etc. In addition, suitable cell lines well known in the art can be obtained from cell line depositories such as the American Type Culture Collection (ATCC).

The host cell of the cell line expressing the target protein according to one embodiment of the present invention may be Chinese hamster ovary cells (CHO), preferably Glutamine Synthetase by recombinant Adeno-Associated Virus (rAAV). HD-BIOP3 cells in which exon 6 of the gene is knocked out can be used.

In order to introduce an expression vector having a genetic sequence encoding a target protein into the host cell, any method for introducing nucleic acid into the cell may be used, and techniques known in the art may be used depending on the host cell. For example, heat shock, electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, and lithium acetate-DMSO method, but are not limited thereto.

Specifically, culturing the cell line expressing the target protein may include culturing the cell line and main-cultivating the cell line.

As used herein, the term "seed culture" refers to culture for the purpose of obtaining a large amount of cell lines. The seed culture may be cultured under temperature conditions that can increase the number of cells most actively. In other words, the cell line may be cultured in order to secure a certain number of cells.

As used herein, the term "production culture" means mass-cultivating a cell line to produce a target protein.

The step of culturing the species may be culturing the cell line expressing the target protein by any one method selected from the group consisting of subculture, suspension culture, and combinations thereof. Preferably, the cell line expressing the target protein can be cultured by suspension culture and subculture.

As used herein, the term "suspension culture" refers to a culture method in which cells are suspended in a culture medium, and cells proliferating in a suspended state in vivo, such as blood cells or multiple cancer cells, are shaken or rotated. Suspension culture can be carried out even if not performed, but in most cases, it can be cultured by rotating the gaiter (gait culture), shaking each culture bottle (shaking culture), or rotating the incubator (swirling culture).

In one embodiment of the present invention, the suspension culture may be carried out under conditions of an agitation speed of 120 RPM to 160 RPM, but is not limited to these conditions. The number of cells inoculated in the suspension culture may be 2x10 ⁵ cells/mL to 5x10 ⁵ cells/mL, 3x10 ⁵ cells/mL to 4x10 ⁵ cells/mL, or 4x10 ⁵ cells/mL to 5x10 ⁵ cells/mL. Not limited.

As used herein, the term "subculture" refers to a method of culturing while transferring a cell line to the same or different fresh medium according to a culture cycle.

In one embodiment of the present invention, the subculture is carried out by transferring the cell line expressing the target protein to the same fresh medium at intervals of 1 to 7 days, 2 to 5 days, 3 to 4 days, or 2 to 3 days. It can be inoculated and cultured. At this time, the number of cells to be inoculated may be 2x10 ⁵ cells/mL to 5x10 ⁵ cells/mL, 3x10 ⁵ cells/mL to 4x10 ⁵ cells/mL, or 4.5x10 ⁵ cells/mL to 5.5x10 ⁵ cells/mL. It is not limited to conditions. In addition, the CO ₂ concentration during subculture may be 4.0% to 6.0%, 4.5% to 5.5%, or 5.0%, but is not limited to these conditions.

Specifically, the seed culturing may be culturing the cell line expressing the target protein at a temperature of 34°C to 38°C. Preferably, the cell line may be cultured at a temperature of 36.5°C.

In one embodiment of the present invention, in the step of culturing the species, the cell line expressing the target protein may be cultured in a medium known to be suitable for cell growth in the art to which the present invention pertains, but is not limited to these conditions. The medium may be CD OptiCHO medium.

In the main culturing step, the seed cultured cell line may be cultured by any one method selected from the group consisting of batch culture, fed-batch culture, continuous culture, and combinations thereof. Preferably, a cell line expressing a target protein may be cultured in a fed-batch culture method.

In one embodiment of the present invention, the step of main culturing the cell line expressing the target protein may be produced by suspension culture. At this time, the suspension culture is 60 RPM to 100 RPM, 65 RPM to 95 RPM, 70 RPM to 95 RPM, 75 RPM to 95 RPM, 80 RPM to 95 RPM, 85 RPM to 95 RPM, 90 RPM to 95 RPM, 92 to It may be carried out under a stirring speed condition of 94 RPM or 93 RPM, but is not limited to these conditions.

In another embodiment of the present invention, the number of cells to be inoculated when culturing the cell line expressing the target protein is 5x10 ⁵ cells/mL to 10x10 ⁵ cells/mL, 6x10 ⁵ cells/mL to 10x10 ⁵ cells/mL, 7x10 ⁵ cells/mL. It may be mL to 10x10 ⁵ cells/mL, 7x10 ⁵ cells/mL to 9x10 ⁵ cells/mL, or 7.2x10 ⁵ cells/mL to 8.8x10 ⁵ cells/mL, but is not limited thereto.

In another embodiment of the present invention, in the step of culturing the cell line expressing the target protein, the pH of the cell line culture medium can be adjusted using a base solution such as carbon dioxide or sodium bicarbonate. Preferably, the pH of the culture medium can be adjusted using carbon dioxide and sodium bicarbonate. More preferably, the pH of the culture medium can be adjusted using carbon dioxide and 8% sodium bicarbonate. In addition, the basic solution introduced to adjust the pH of the culture solution during culture maintains the pH of the culture solution at an appropriate level by being present in the culture solution in an appropriate ratio with lactic acid secreted by the cells in the culture solution. At this time, the pH of the culture medium (deadband, error range is 0.05) can be adjusted to 6.8 to 7.5, 6.9 to 7.4, 7.0 to 7.3, 7.0 to 7.4, 6.8 to 7.2 or 6.8 to 7.1. Preferably, the pH of the culture medium can be adjusted to pH 6.8 to 7.1 (deadband, which is an error range, is 0.05).

In this way, in adjusting the culture solution to be maintained at a pH within a certain numerical range, at this time, as the upper limit of the pH value range (hereinafter referred to as "upper pH value") is lowered, the amount of carbon dioxide injected into the culture solution in the latter half of the culture is reduced and the formation of bubbles in the culture solution is prevented. effect appears. As the culture progresses, a phenomenon in which the lactic acid content in the culture medium decreases and the pH of the culture medium rises, and this pH rise causes a large amount of carbon dioxide to be injected through a sparger located at the bottom of the bioreactor. In addition, since the injector plays a role of introducing carbon dioxide and oxygen into the culture medium, the transferability of oxygen into the culture medium decreases due to an increase in the ratio of carbon dioxide/oxygen in the air introduced into the culture medium as the pH rises. Reduction in oxygen delivery power to the culture medium increases the frequency of oxygen injection through the injector by reducing the amount of dissolved oxygen in the culture medium (Dissolved Oxygen, DO). However, direct injection of gas into the culture medium may be a factor in increasing the pH of the culture medium.

On the other hand, in the process of continuing the culture, the increase in pH of the culture medium due to the decrease in lactic acid in the culture medium interacts with the decrease in secretion of lactic acid secreted by the cells in the culture medium. An increase in pH in the culture medium reduces the secretion of lactic acid from the cells, which in turn causes a vicious cycle in which the pH of the culture medium increases. Have no choice but to. Therefore, in adjusting the pH in the culture medium, if the upper pH value is set as low as 7.15 or less, 7.10 or less, 7.05 or less, 7.00 or less, 6.95 or less, 6.90 or less, 6.85 or less, or 6.80 or less, carbon dioxide and Oxygen consumption can be reduced.

In one embodiment of the present invention, compared to the case where the pH in the culture medium of the present invention is 7.25, if the pH in the culture medium is 6.8 to 7.1 (deadband of the error range is 0.05), carbon dioxide, oxygen, and hydrogen carbonate introduced into the culture medium during the culture period Production costs can be reduced by reducing the amount of basic substances such as sodium.

In one embodiment of the present invention, by maintaining the pH in the culture medium at 6.8 to 7.1 (the deadband, which is the error range, is 0.05), the amount of sodium bicarbonate introduced into the culture medium at the end of the culture is higher than the amount of sodium bicarbonate introduced into the culture medium before the culture. can reduce At this time, the reference point for distinguishing the early and late stages of culture may be the 4th day or the 5th day of culture.

In one embodiment of the present invention, the air sparging rate of the gas (meaning general air constituting the earth's atmosphere) sprayed through the injector during the culturing period may be changed.

In another embodiment of the present invention, the gas injection rate in the early stage of culture and the gas injection rate in the later stage of culture may be different. Preferably, the gas ejection rate in the late stage of culture may be half or less of the gas ejection rate in the early stage of culture. More preferably, the gas ejection rate after the 4th or 5th day of culture may be half or less of the gas ejection rate before the 4th or 5th day of culture.

In another embodiment of the present invention, the gas injection rate is 0.0005 to 0.01 vvm (volume air/volume media/minute), 0.0005 to 0.009 vvm, 0.0005 to 0.008 vvm at the late stage of culture, preferably after the 4th or 5th day of culture. .

In one embodiment of the present invention, DO in a culture medium of a cell line expressing a target protein may be maintained within a certain range. At this time, the DO of the cell line culture may be maintained at 10% to 90%, 20% to 80%, 30% to 70%, 40% to 60%, 40% to 50% or 40%, but is not limited to these conditions. don't

In another embodiment of the present invention, when the DO of the cell line culture medium is located at the lower end of the preferred DO level range of the culture medium, the DO level of the culture medium may be maintained by supplying oxygen to the culture medium.

In one embodiment of the present invention, the flow rate of oxygen to the bioreactor during the culturing period may be maintained at 20 LPM or less.

In another embodiment of the present invention, the carbon dioxide flow rate into the bioreactor may be maintained at 5 LPM or less at the later stage of culture, preferably after the 4th or 5th day of culture.

In one embodiment of the present invention, a cell line expressing a target protein may be cultured at a temperature of 30°C to 38°C.

In another embodiment of the present invention, the culture temperature at the end of culture, preferably after the 4th or 5th day of culture, may be lower than the culture temperature before the culture, preferably before the 4th or 5th day of culture.

In another embodiment of the present invention, a cell line expressing a target protein may be cultured at a temperature of 37° C. for a certain period of time at the beginning of culture, and then cultured at a lower temperature. Preferably, the cell line is cultured at a temperature of 37 ° C until the 4th or 5th day of culture, and after the 5th or 6th day of culture, the temperature is 1 ° C, 2 ° C, 3 ° C, 4 ° C, 5 ° C, 6 ° C, 7 ° C. C, 8 ℃, 9 ℃ or 10 ℃ lower temperature can be incubated. More preferably, the cell line may be cultured at a temperature of 37 ° C. until the 4th to 5th day of culture, and then cultured at a temperature of 31 ° C. during the culture period.

In another embodiment of the present invention, a cell line expressing a protein of interest can be cultured in a medium known in the art to be suitable for cell growth, but is not limited to these conditions. The medium may be Dynamis medium.

In another embodiment of the present invention, it may be treated with an antifoaming agent for the purpose of removing bubbles generated in the culture medium. The antifoaming agent can be used by selecting a suitable material in consideration of the components of the culture medium, and is used when bubbles reach the bottom of the air filter in the bioreactor. Preferably, the antifoaming agent may be an ADCF antifoaming agent.

In one embodiment of the present invention, a cell line expressing a target protein may be cultured in a flask or bioreactor. Unlike flasks, culture using a bioreactor can control and maintain culture conditions such as DO, glucose content, and pH, so that cell culture can be performed under favorable conditions and mass culture is possible.

The types of flasks and bioreactors and culture conditions can be changed within a range commonly controllable by those skilled in the art.

For example, a cell line expressing a target protein is inoculated into an Elenmeyer flask, cultured in suspension, subcultured to secure the minimum number of cells to be inoculated into a bioreactor, and the subcultured cell line is equipped with a disposable cell culture bag. The target protein can be produced by inoculating the prepared bioreactor and performing fed-batch culture.

In another embodiment of the invention, the culture period may be 6 days or more. Preferably, the culture period is 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days or 20 days. can More preferably, the culture period may be 10 days.

Example

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only for exemplifying the present invention, and the scope of the present invention is not limited only to these.

Preparation Example 1. Preparation of cell lines expressing the target protein

To prepare a cell line expressing the polypeptide represented by the amino acid sequence of SEQ ID NO: 1, HD-BIOP3 cell line obtained from HORIZON Discovery, UK was used as a host cell. A recombinant expression vector plasmid (M-2560) was constructed by inserting the cDNA synthesized by Genscript into the Donor plasmid (pJV145) using the SalI/NotI restriction enzyme reaction (Fig. 1). The M-2560 vector has a DNA sequence represented by SEQ ID NO: 3.

A recombinant expression vector plasmid (M-2560) was transfected into the HD-BIOP3 cell line. Among the transfected cells, cells capable of stable single cell cloning were selected to prepare a research cell bank (RCB) having sufficient long-term passage stability. The RCB was transferred to the Andong plant of SK Bioscience, and a working cell bank (WCB) was manufactured in accordance with GMP standards.

One vial of WCB (Working Cell Bank) was dissolved in a water bath at 37 ° C., diluted with CD OptiCHO medium, centrifuged, and the pellet was resuspended in CD OptiCHO medium. 6 L culture at 5 x 10 ⁵ cells/mL in 10 L bioreactor, 30 L culture at 5 x 10 ⁵ cells/mL in 50 L bioreactor, 5 x 10 ⁵ cells/mL in 200 L bioreactor 200 L culture, 8 x 10 ⁵ cells / mL concentration in a 2000 L bioreactor was expanded and subcultured so that it could be cultured at 1600 L.

Production Example 2. Cultivation of a cell line expressing a target protein in a 2000 L bioreactor

After mounting the disposable cell culture bag in a 2000 L bioreactor and preparing for operation, 1400 L of Dynamis medium was injected, and WCB was inoculated at a concentration of 8.0 x 10 ⁵ cells/mL and cultured. During incubation, DO control proceeded through gas and oxygen, and pH control proceeded using 8% sodium bicarbonate and carbon dioxide gas.

Sampling was performed every day immediately after inoculation to measure the number of cells, check the presence of microbial contamination and the concentration of glucose in the culture medium, and when bubbles in the bioreactor reached the bottom of the air filter mounted on the condenser, ADCF antifoam was treated. This process was repeated until the end of the culture. Culture conditions used during culture are shown in Table 1 below.

Cell line culture conditions culture conditions set value Temperature (day 0-5 of incubation) 37℃ Temperature (after the 5th day of culture) 31℃ culture medium Dynamis medium for inoculation CD OptiCHO stirring speed 93 RPM culture volume 1600L initial inoculation concentration 8.0 x 10 ⁵ cells/mL amount of dissolved oxygen 40% Dissolved Oxygen Control gas, oxygen pH control 8% sodium bicarbonate, carbon dioxide

Experimental Example 1. Change according to pH change in the culture medium in the bioreactor

In Preparation Example 2, as the upper pH value of the culture solution in the bioreactor reached 7.25 on the 6th day of culture, the injection of carbon dioxide into the culture solution through the sprayer increased and the injection of oxygen to maintain DO in the culture solution increased for pH control. occurred (FIG. 2). In addition, there is a problem in that the generation of bubbles increases as the injection of carbon dioxide and oxygen increases.

Therefore, it was verified whether the above problem could be solved by setting the upper pH value of the culture medium in the bioreactor to pH 7.1 (the deadband, which is the pH error range, is 0.05).

Specifically, when the upper pH value of the culture medium in the bioreactor is set to pH 7.2 (deadband is 0.05) (CTIA01503 batch) and when the upper pH value of the culture medium is set to pH 7.1 (deadband is 0.05) (batch E21R054) Changes over the period were observed. Even after the 4th or 5th day of culture, the E21R054 batch did not show an increase in the amount of oxygen and carbon dioxide injected into the culture medium, and as the pH was maintained at a constant level, the total amount of sodium bicarbonate accumulated and used during the culture period increased. found to be low (Fig. 3).

In addition, when the upper pH value of the culture medium in the bioreactor was set to pH 6.8, 6.99, 7.0 or 7.2, the lower the pH, the lower the amount of lactic acid produced during the culture period (FIG. 4). Therefore, by keeping the upper pH value low during the culture period, the cells do not forcibly produce by-products such as lactic acid to maintain the pH. When the concentration of lactic acid in the culture medium increases, the amount of buffer input such as sodium bicarbonate, which is separately added to maintain the pH of the culture medium, decreases. In addition, the stress experienced by the cells during the culture process was reduced, and the culture stability was greatly improved, resulting in increased protein production efficiency.

Experimental Example 2. Confirmation of the foam suppression effect by changing the gas injection speed during the culture process

During cultivation in a bioreactor, gas forming bubbles is generated in the culture medium as agitation is performed, which may adversely affect bacterial growth in the culture medium and cell protein expression. Therefore, antifoams are added to the culture medium for the purpose of inhibiting the formation of bubbles in the culture medium, but the antifoams cause cell membrane permeability changes to inhibit cell growth and affect protein expression. It was verified whether foam formation generated in the culture medium could be suppressed by changing the injection speed of the gas introduced into the culture medium.

Considering the upper pH value of pH 7.1 (deadband is 0.05) of the culture medium confirmed to increase the production efficiency in Experimental Example 1, culturing under the conditions shown in Table 2 below while maintaining the pH of the culture medium at pH 6.8 to 7.1. The effect of suppressing bubbles generated in the culture medium was confirmed.

process conditions incubation period DO output§ Air sparging flow (vvm) degree of foaming
(1-5) control example Day 0 to Day 10 0% 0 5 10% 0.002 100% Example 1 Day 0 to Day 5 0% 0.002 3 10% 100% Day 5 to Day 10 0% 0.0005 One 10% 100% Example 2 Day 0 to Day 5 0% 0.002 3 10% 100% Day 5 to Day 10 0% 0.001 One 10% 100%

§DO output: As an indicator of the amount of oxygen that needs to be replenished. If DO ouput is 0%, it means that dissolved oxygen is sufficient in the culture medium, and if it is 100%, it means that all dissolved oxygen is consumed in the culture medium. do.

In the case of the control example in which gas was not sprayed when there was no need to replenish oxygen, and gas was sprayed at a constant spray speed in other cases, the degree of foaming in the culture medium was the highest.

On the other hand, in Examples 1 and 2 in which the gas injection rate was reduced after the 5th day of culture regardless of DO output, foam formation was found to be suppressed. In addition, in Examples 1 and 2, process convenience was ensured because gas was sprayed at the same spray rate regardless of the DO output value before and after the 5th day of culture.

On the other hand, in Example 1, a slight pH decrease was observed to the upper pH deadband level in the second half of the culture, but it was shown that the yield decreased slightly. Unlike Example 1, Example 2 did not cause a decrease in pH in the latter part of the culture, and it was shown that the yield was maintained.

<110> SK bioscience Co., Ltd. <120> METHOD FOR CULTURING A CELL EXPRESSING A PROTEIN <130> FDP/202110-0043 <150> KR 10-2021-0137835 <151> 2021-10-15 <160> 3 <170> KoPatentIn 3.0 <210> 1 < 211> 467 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 1 Met Gly Ile Leu Pro Ser Pro Gly Met Pro Ala Leu Leu Ser Leu Val 1 5 10 15 Ser Leu Leu Ser Val Leu Leu Met Gly Cys Val Ala Glu Thr Gly Thr 20 25 30 Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn 35 40 45 Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser 50 55 60 Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser 65 70 75 80 Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys 85 90 95 Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val 100 105 110 Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr 115 120 125 Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn 130 135 140 Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu 145 150 155 160 Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu 165 170 175 Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn 180 185 190 Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val 195 200 205 Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His 210 215 220 Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Gly Gly Ser Gly 225 230 235 240 Gly Ser Gly Ser Gly Gly Ser Gly Gly Ser Gly Ser Glu Lys Ala Ala 245 250 255 Lys Ala Glu Glu Ala Ala Arg Lys Met Glu Leu Phe Lys Lys His 260 265 270 Lys Ile Val Ala Val Leu Arg Ala Asn Ser Val Glu Glu Ala Ile Glu 275 280 285 Lys Ala Val Ala Val Phe Ala Gly Gly Val His Leu Ile Glu Ile Thr 290 295 300 Phe Thr Val Pro Asp Ala Asp Thr Val Ile Lys Ala Leu Ser Val Leu 305 310 315 320 Lys Glu Lys Gly Ala Ile Ile Gly Ala Gly Thr Val Thr Ser Val Glu 325 330 335 Gln Ala Arg Lys Ala Val Glu Ser Gly Ala Glu Phe Ile Val Ser Pro 340 345 350 His Leu Asp Glu Glu Ile Ser Gln Phe Ala Lys Glu Lys Gly Val Phe 355 360 365 Tyr Met Pro Gly Val Met Thr Pro Thr Glu Leu Val Lys Ala Met Lys 370 375 380 Leu Gly His Thr Ile Leu Lys Leu Phe Pro Gly Glu Val Val Gly Pro 385 390 395 400 Gln Phe Val Lys Ala Met Lys Gly Pro Phe Pro Asn Val Lys Phe Val 405 410 415 Pro Thr Gly Val Asn Leu Asp Asn Val Ala Glu Trp Phe Lys Ala 420 425 430 Gly Val Leu Ala Val Gly Val Gly Ser Ala Leu Val Lys Gly Thr Pro 435 440 445 Asp Glu Val Arg Glu Lys Ala Lys Ala Phe Val Glu Lys Ile Arg Gly 450 455 460 Ala Thr Glu 465 <210> 2 <211> 1404 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence <400> 2 atgggaatcc tgccaagccc tggaatgcca gccctgctgt ccctggtgtc tctgctgagc 60 gtgctgctga tgggatgcgt ggcagagacc ggaacaaggt tccctaacat caccaacctg 120 tgcccattcg ct gcgaggtccgt gtatgc ctggaaccgg 180 aagagaatct ctaattgcgt ggccgactat agcgtgctgt acaatagcgc ctccttctct 240 acctttaagt gctatggcgt gtctcccacc aagctgaacg acctgtgctt cacaaacgtg 300 tacgccgaca gctttgtgat ccggggcgat gaggtgagac agatcgcacc aggacagacc 360 ggcaagatcg cagactacaa ctataagctg cctgacgatt tcacaggctg cgtgatcgcc 420 tggaatagca acaatctgga ttccaaagtg ggcggcaact acaattatct gtacagaggattcctgacagaggcctgacca gattgactg 480 catca gcaccgagat ctaccaggca 540 ggctccacac catgcaacgg agtggagggc ttcaattgtt attttcccct gcagagctac 600 ggcttccagc ctaccaatgg cgtgggctat cagccataca gagtggtggt gctgtcctttt 660 gagctgctgc acgcaccagc aaccgtgggc20 ggaccgtgggc agtaca ggat ccggaggatc cggaggatct ggaagcgaga aggcagcaaa ggcagaggag 780 gcagcaagga agatggagga gctgttcaag aagcacaaga tcgtggccgt gctgagagcc 840 aactctgtgg aggaggccat cgagaaggca gtggccgtgt tcgcaggagg agtgcacctg 900 atcgagatca cctttacagt gcccggacc gcccggacc gtgctg 960 aaggagaagg gagcaatcat cggagcagga accgtgacat ctgtggagca ggcaaggaag 1020 gcagtggagt ccggagccga gtttatcgtg tctcctcacc tggatgagga gatctcccag 1080 ttcgccaagg agaagggcgt gttttacatg cctggcgtga 1gacctgaggtga tgacctgagcaacg aga1 gggcca caccatcctg aagctgttcc caggagaggt ggtgggacca 1200 cagtttgtga aggccatgaa gggcccattc cccaatgtga agtttgtgcc tacaggcggc 1260 gtgaacctgg acaatgtggc agagtggttc aaggcaggcg tgctggcagt gggagtggga 1320 tctgccccctgg acaccagaggagggac c ctttgtggag 1380 aagatcaggg gagcaacaga gtga 1404 <210> 3 <211> 10704 <212> DNA <213> Artificial Sequence <220> < 223>Artificial Sequence <400> 3 gc 180 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300 tacgccagct ggcgaaaggg ggatgtgctg caaggcgggta 300 tacgccagct tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ggagatcggt acttcgcgaa 420 tgcgtcgaga tgtttaaact cccgccccta actccgccca gttccgccca ttctccgccc 480 catggctgac taattttttt tatttatgca gaggccgagggt ccgcctcggtt ctgagagaggtt cttttttgga ggcctaggct tttgcaaaaa gctagctggt 600 tctttccgcc tcagaaggta cctaaccaag ttcctctttc agaggttatt tcaggccacc 660 ttccaccatg gccacctcag caagttccca cttgaacaa aacatcaagc aaatgtactt 720 aaatgtactt c catgtatatc tgggttgatg gtactggaga 780 aggactgcgc tgcaaaaccc gcaccctgga ctgtgagccc aagtgtgtag aagagttacc 840 tgagtggaat tttgatggct ctagtacctt tcagtctgag ggctccaaca gtgacatgta 900 tctcagccct gttgccatgt ttcgggaccc cttccgcaga gatcccaaca agctggtcct cttggaagtgtt cttggaagtgtt 960 tgcagagacc aatttaaggc actcgtgtaa 1020 acggataatg gacatggtga gcaaccagca cccctggttt ggaatggaac aggagtatac 1080 tctgatggga acagatgggc acccttttgg ttggccttcc aatggctttc ctgggcccca 1140 caggtccggtat tactgtacggggg tgg tcgtggaggc 1200 tcactaccgc gcctgcttgt atgctggggt caagattaca ggaacaaatg ctgaggtcat 1260 gcctgcccag tgggaatttc aaataggacc ctgtgaagga atccgcatgg gagatcatct 1320 ctgggtggcc cgtttcatct tgcatcgagt atgtgaagac tttggggtaa tagcaacctt 1380 tgaccccaag cccatttgccagccctg ggaacttgccagctg a actttagcac 1440 caaggccatg cgggaggaga atggtctgaa gcacatcgag gaggccatcg agaaactaag 1500 caagcggcac cggtaccaca ttcgagccta cgatcccaag gggggcctgg acaatgcccg 1560 tcgtctgact gggttccacg aaacgtccaa catcaacgac ctg6cc tgt0 tcgcagtgcc agcatccgca ttccccggac tgtcggccag gagaagaaag gttactttga 1680 agaccgccgc ccctctgcca attgtgaccc ctttgcagtg acagaagcca tcgtccgcac 1740 atgccttctc aatgagactg gcgacgagcc cttccaatac aaaaactaac gcccgcccca 1800 cgacccgcag cgcccgaccg cgcaca catcataaga 1860 tacattgatg agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt 1920 gaaatttgg atgctattgc tttattgta accattataa gctgcaataa acaagttaac 1980 aacaacaatt gcattcattt tatgtttcag gttcag0gta agttcag0gatta aggtcag0gatta aa acctctacaa atgtggtaga attctacgta gataaaagtt ttgttacttt 2100 atagaagaaa ttttgagttt ttgttttttt taataaataa ataaacataa ataaattgtt 2160 tgttgaattt attattagta tgtaagtgta aatataataa aacttaatat ctattcaaat 2220 taataaataa acctcgatacat cagaattcattg2 cagaattcatg2 0 atctttaacg tacgtcacaa tatgattatc tttctagggt taatctagct gcgtgttctg 2340 cagcgtgtcg agcatcttca tctgctccat cacgctgtaa aacacatttg caccgcgagt 2400 ctgcccgtcc tccacgggtt caaaaacgtg aatgaacgag gcgcgctcat atcatcat ggg taactcacgg ggtatccatg tccatttctg cggcatccag 2520 ccaggatacc cgtcctcgct gacgtaatat cccagcgccg caccgctgtc attaatctgc 2580 acaccggcac ggcagttcca tttaaatggc tgtcgccggt attgttcggg ttgctgatgc 2640 gcttcgggct gaccatccgg aactgtgtcc c70accgagccg tggcctgaac gaacagttca ccgttaaagg cgtgcatggc cacaccttcc cgaatcatca 2760 tggtaaacgt gcgttttcgc tcaacgtcaa tgcagcagca gtcatcctcg gcaaactctt 2820 tccatgccgc ttcaacctcg cgggaaaagg cacgggctcagtc gtccc8cttc acc ct tgggcgatga ctgagccgga aaaaagaccc gacgatatga tcctgatgca 2940 gctagattaa ccctagaaag atagtctgcg taaaattgac gcatgcattc ttgaaatatt 3000 gctctctctt tctaaatagc gcgaatccgt cgctgtgcat ttaggacatc tcagtcgccg 3060 cttggagctc ccgtgaggcg tgcttgtcaa tgcttgtcaa tgcttgtcaa tgcggtaagt tgt1actgg gt gagtcaaaat gacgcatgat tatcttttac gtgactttta agatttaact 3180 catacgataa ttatattgtt atttcatgtt ctacttacgt gataacttat tatatatata 3240 ttttcttgtt atagatatca agcttataga tctggggaca gccccccccc aaagccccccc aaagccccccc aaagcccagctcgg ccgcccagctcgg ccgccgctccc tacgt ccgcccg gggctccgct 3360 ccggtccggc gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg 3420 gggaaggtgg cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag 3480 acacctgggg ggatacgggg aagtaaagcttt aggctgaaag agagatttag 3gagggacaga gt g caaaggagca cagtgctcat ccagatccaa ccccctgcta 3600 tgtgcagggt catcaaccag cagcccaggc tgcccagagc cacatccagc ctggccttga 3660 atgcctgcag ggatggggca tccacagcct ccttgggcaa cctgttcagt gcgtcaccactcatac 3720 acctactggggggg aaac ctcccctgtc tcagtgtaaa 3780 gccattcccc cttgtcctat caagggggag tttgctgtga cattgttggt ctggggtgac 3840 acatgtttgc caattcagtg catcacggag aggcagattt ggggataagg aagtgcagga 3900 cagcatggac gtgggacatg caggtgttga gggctctggg acactctcca agtcacgatagagagaccagcg 3960 agtcagatagagaggaccta caaagagcaa gttaaaaccc 4020 agcatggaga ggagcacaaa aaggccacag acactgctgg tccctgtgtc tgagcctgca 4080 tgtttgatgg tgtctggatg caagcagaag gggtggaaga gcttgcctgg agagatacag 4140 ctgggtcagt aggactgggt0 caggatcagtcc at2 gagatcagctg gagatcagctg gagatcagctg gtcaaatcat gaaggctgga aaagccctcc aagatcccca agaccaaccc caacccaccc 4260 accgtgccca ctggccatgt ccctcagtgc cacatcccca cagttcttca tcacctccag 4320 ggacggtgac ccccccacct ccgtgggcag ctgtgccact gcagcaccgc tctttggaga 4380 aggctaaatct cc aggctaaatct tgctaaatct tgctaaatct tgctaaatctcc ca caacgtaagg ccattatctc 4440 tcatccaact ccaggacgga gtcagtgagg atggggctct agagtcaaca ggaaagttcc 4500 attggagcca agtacattga gtcaataggg actttccaat gggttttgcc cagtacataa 4560 ggtcaatggg aggtaagcca atgggttaggac g 6c cattatactg g tgaccattactg 4c ttcc aatgggtttt gcccagtaca taaggtcaat aggggtgaat caacaggaaa 4680 gtcccattgg agccaagtac actgagtcaa tagggacttt ccattgggtt ttgcccagta 4740 caaaaggtca atagggggtg agtcaatggg tttttcccat tattggcacg tacataaggt 4800 caataggggt gagtacgttacca agttcagtttacca ttcc 4860 caccattgac gtcaatgggc tattgaaact aatgcaacgt gacctttaaa cggtactttc 4920 ccatagctga ttaatgggaa agtaccgttc tcgagccaat acacgtcaat gggaagtgaa 4980 agggcagcca aaacgtaaca ccgccccggt tttcccctggtaattcatt4cata5 ggc tgagctgcgt tctacgtggg tatataagca gagctctccc tatcagtgat 5100 agagatctcc ctatcagtga tagagatcga gctcagcgtc ggtaccgtac ctcttccgca 5160 tcgctgtctg cgagggccag ctgttggggt gagtggcggg tgtggcttcc gcgggccccg 5220 gagctggaggtc cctgctctga gcgggtgggtcg cc gcgggccgtcgg ct 5280 gctgtgagca ttcccacttc gagtggcggg cggtgcgggg gtgagagtgc gaggcctagc 5340 ggcaaccccg tagcctcgcc tcgtgtccgg cttgaggcct agcgtggtgt ccgccgccgc 5400 gtgccactcc ggccgcacta tgcagttttttt ggtcctct4cc ggcagttttttct ggtccctttgct 60 gcatgggcta acaaagggag ggtgtggggc tcactcttaa ggagcccatg aagcttacgt 5520 tggataggaa tggaagggca ggaggggcga ctggggcccg cccgccttcg gagcacatgt 5580 ccgacgccac ctggatgggg cgaggcctgt ggctttccga agcaatcggg cgtgagttta 5640 gcctacctgg gccatgtggc cctagcactg ggtgccgggcgt ggtgccgggcgt 0 0 ccttgcctcc caacaagggt gaggccgtcc cgcccggcac cagttgcttg cgcggaaaga 5760 tggccgctcc cggggccctg ttgcaaggag ctcaaaatgg aggacgcggc agcccggtgg 5820 agcgggcggg tgagtcaccc acacaaagga agagggcct8gcc tgagtcaccc acacaaagga agagggcct8gcc tggccgtct5 gacc ccgtggtcta tcggccgcat agtcacctcg ggcttctctt gagcaccgct 5940 cgtcgcggcg gggggagggg atctaatggc gttggagttt gttcacattt ggtgggtgga 6000 gactagtcag gccagcctgg cgctggaagt cattcttgga atttgcccct ttgagtttgg 6060 agcgaggcta attctcaagc ctcttagcgg6tacta 0 ctcgcggttg aggacaaact cttcgcggtc tttccagtac tcttggatcg gaaacccgtc 6180 ggcctccgaa cggtactccg ccaccgaggg acctgagcga gtccgcatcg accggatcgg 6240 aaaacctcgt cgacgccgcc accatgggaa tcctgccag30gc ggt gtctctgctg agcgtgctgc tgatgggatg cgtggcagag accggaacaa 6360 ggttccctaa catcaccaac ctgtgcccat tcggcgaggt gtttaacgcc acacgctttg 6420 cctccgtgta tgcctggaac cggaagagaa tctctaattg cgtggccgac tatagcgtgc 6480 tgtacaatag cgcctccttc tctaccttta agtgctat0 tgcctggaac acctcagctgacct5 accaagctgaaccct gtgtacgccg acagctttgt gatccggggc gatgaggtga 6600 gacagatcgc accaggacag accggcaaga tcgcagacta caactataag ctgcctgacg 6660 atttcacagg ctgcgtgatc gcctggaata gcaacaatct ggattccaaa gtgggcggca 6720 gagtcagcggca 6720 gagtcagcggacta ctgagtcagtccacacta gagcggggaca 6780 tcagcaccga gatctaccag gcaggctcca caccatgcaa cggagtggag ggcttcaatt 6840 ggtattttcc cctgcagagc tacggcttcc agcctaccaa tggcgtgggc tatcagccat 6900 acagagtggt ggtgctgtcc tttgagctgc tgcacgcacc agcaaccgtg tgcggacctg tgcggacc0 gaag6 aggaagcg gatccggagg atccggagga tctggaagcg 7020 agaaggcagc aaaggcagag gaggcagcaa ggaagatgga ggagctgttc aagaagcaca 7080 agatcgtggc cgtgctgaga gccaactctg tggaggaggc catcgagaag gcagtcgccag aggctagtcagcagg aggctag gtgcccgac gccgataccg 7200 tgatcaaggc cctgtccgtg ctgaaggaga agggagcaat catcggagca ggaaccgtga 7260 catctgtgga gcaggcaagg aaggcagtgg agtccggagc cgagtttatc gtgtctcctc 7320 acctggatga ggagatctcc cagttcgcca aggagaaggg cgtgttttac atgcctggcg 7380 tgatgacccc aacagagctggcg ctgaagctgt 7440 tcccaggaga ggtggtggga ccacagtttg tgaaggccat gaagggccca ttccccaatg 7500 tgaagtttgt gcctacaggc ggcgtgaacc tggacaatgt ggcagagtgg ttcaaggcag 7560 gcgtgctggc agtgggagtg ggatctggagg gggacctgacccgg gggacctgaccc 620 aaaaggccaa ggcctttgtg gagaagatca ggggagcaac agagtgagcg gccgccacac 7680 atcataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc 7740 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa 7800 caagttaaca gaattcaat gatttggtcat at gag 7860 gttttttaaa gcaagtaaaa cctctacaaa tgtggtacac aaagtgggga gctctagagg 7920 gacagccccc ccccaaagcc cccagggatg taattacgtc cctcccccgc tagggggcag 7980 cagcgagccg cccggggctc cgctccggtc cggcgctccc c4gccggggccc c4gccggggccc cgccgggccc8 cccggg cacggggaag gtggcacggg atcgctttcc tctgaacgct 8100 tctcgctgct ctttgagcct gcagacacct ggggggatac ggggaaaaag gggagctcta 8160 gagggacagc ccccccccaa agcccccagg gatgtaatta cgtccctccc ccgctagggg 8220 gcagcagcga gccgcccgc gctccccgccgc gctccccgccgc ccccgagcc 8280 ggcagcgtgc ggggacagcc cgggcacggg gaaggtggca cgggatcgct ttcctctgaa 8340 cgcttctcgc tgctctttga gcctgcagac acctgggggg atacgggggaa aaaggatcca 8400 tggccggcca tctgcagatc atatgatgggacgg atgcc agcc 8460 aagcgagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 8520 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 8580 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 8640 cgtgccagct gcattaatga atcggccaac gcgcattgggt gcgcattgggt agggt8 cttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 8760 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 8820 agaacatgtg agcaaaaggc cagcaaaagg0 cagcaaaagg0 ccaggaaggt ccaggaaggtcc gtaata8ggtcc taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 8940 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 9000 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 9060 gaagcgtggc gctttctcat agctcacgct gtaggtattggt gcagt120 ctggtc tag9 ccaagct gggctgtgg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 9180 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 9240 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagt aggt 9240 tatttggtat ctgcgctctg ctgaagccag 9360 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 9420 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 9480 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 9540 tggtcatgag attatcaaaa aggatcttca cctagatcct ttaatagaattataa ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca 9660 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg 9720 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatcaccact gcaatgactgatac 9780 cgctgactgatacg at aaaccagcca gccggaaggg 9840 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc 9900 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta 9960 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc gccatt2cag0 gccgtcaggaggt cccatgt tgtgcaaaaa agcggttagc tccttcggtc 10080 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac 10140 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtcagagtc ggtacagagt0 ggtacagagt0 ggtacaggtact gaccgag ttgctcttgc ccggcgtcaa 10260 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt 10320 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca 10380 ctcggcacc caactgatct tcagcatctt ttactttcac cagcgtttct ggggaacgagcaa 10440 aggaacagagcaa gaataagggc gacacggaaa tgttgaatac 10500 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg 10560 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc 10620 gaacatatagtgcc acctgacgttaac tatta ata 10680ggcgtatcac gaggcccttt cgtc 10704

Claims (12)

A method for culturing a cell line expressing a target protein, which is a polypeptide represented by the amino acid sequence represented by SEQ ID NO: 1 or a polypeptide having 75% or more sequence homology thereto, comprising culturing a cell line expressing the protein; , The step of culturing the cell line comprises adjusting the pH of the culture medium, the culture method. The method of claim 1, wherein the cell line is derived from Chinese Hamster Ovary (CHO) cells. The culture method according to claim 1, wherein the pH of the culture medium is adjusted using carbon dioxide and/or sodium bicarbonate. The culture method according to claim 1, wherein the pH of the culture medium is maintained at 6.75 to 7.15. The culture method according to claim 1, wherein the amount of sodium bicarbonate introduced into the culture medium at the later stage of the culture is lower than the amount of sodium bicarbonate introduced into the culture medium prior to the culture. The culture method according to claim 1, wherein an air sparging rate introduced into the culture medium in the late stage of the culture is less than half of the gas sparging rate in the early stage of the culture. The culture method according to claim 6, wherein the gas injection rate in the late stage of the culture is 0.0005 to 0.001 vvm. The culture method according to claim 1, wherein the flow rate of oxygen to the bioreactor in which the culture is performed during the culture period is maintained at 20 LPM or less. The culture method according to claim 1, wherein the flow rate of carbon dioxide to the bioreactor in which the culture is performed in the late stage of culture is maintained at 5 LPM or less. The culture method according to claim 1, wherein the culture temperature in the late stage of culture is lower than the culture temperature in the early stage of culture. The culture method according to claim 10, wherein the culture temperature in the late stage of culture is 1 °C to 10 °C lower than the culture temperature in the early stage of culture. The culture method according to claim 1, wherein the culture period is 6 to 15 days.

Images