Method for producing a fusion protein having an IgG Fc domain

Abstract

To provide a method capable of preventing complication of a safety problem such as immunogenicity and complication of a purification step in culture of animal cells, by suppressing increase of a protein production amount, suppressing generation of misfolded / coagulated protein, suppressing generation of a deamidation body of a protein and a substitution / deficiency body of an amino acid through adjustment and optimization of a cell culture condition related to protein production.SOLUTION: The invention relates to a production method of a fusion protein comprising an IgG Fc domain, in particular, the invention is configured so that, in a production method of a fusion protein comprising the IgG Fc domain, a step for culturing cells which produce the fusion protein at a lowered culture temperature, is added, for increasing cell growth and cell survival rate, for increasing productivity of the fusion protein and suppressing generation of an aggregate, and for improving quality and production yield.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application claims and benefits from priority granted by Korean Patent Application No. 10-2015-0144330, filed on 15 October 2015, and Korean Patent Application No. 10-2016-0132633, filed on 13 October 2016, the entirety of which is incorporated herein by reference. [Background technology]

[0002] The present invention relates to a method for producing a fusion protein having a human immunoglobulin G (IgG)Fc domain, particularly a protein in which the soluble extracellular domain of a vascular endothelial growth factor (VEGF) receptor is fused with a human immunoglobulin G (IgG)Fc domain (e.g., aflibercept).

[0003] Vascular endothelial growth factor (VEGF) is an important factor that increases angiogenesis and vascular permeability. In particular, VEGF is overexpressed in tumor cells and promotes abnormal angiogenesis and tumor growth (Oncogene, 2004, 23, 1745-1753). Abnormal angiogenesis has also been reported to be importantly related to other diseases in addition to tumor development. Abnormal angiogenesis mediated by VEGF is associated with ophthalmic diseases such as exudative macular degeneration, diabetic retinopathy, and macular edema associated with retinal vein occlusion (J. Korean Med. Assoc., 2014, 57, 7, 614-623).

[0004] For treating such ophthalmic diseases, pegaptanib (RNA aptamer), ranibizumab (monoclonal IgG antibody fragment (Fab)), and bevacizumab (monoclonal IgG antibody) are used, and aflibercept (VEGFR1 and VEGFR2 fused with IgG1 Fc) was approved in the United States in 2011 as a treatment for exudative macular degeneration (Biol.Ther., 2012.2.3.1-22; Drug Design Development Therapy, 2013,3,7,711-722).

[0005] Due to the increasing demand for such recombinant therapeutic proteins, much research is being conducted to improve cell growth, viability, and protein production and quality through improvements in cell selection, culture medium optimization, and control of the culture process. Many proteins and polypeptides produced in cell culture are manufactured and separated by culturing cells at a constant temperature and pH for a set period of time using batch or fed-batch methods. Therefore, the yield and quality are affected by the cell culture conditions. There is a need for methods that can increase protein yield, suppress the formation of misfolded / aggregated proteins, and prevent the generation of deamided proteins and amino acid substitutions / deletions, thereby preventing safety issues such as immunogenicity and complicating the purification process in animal cell culture, through the adjustment and optimization of cell culture conditions in relation to protein production. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Oncogene, 2004, 23, 1745-1753 [Non-Patent Document 2] J.Korean Med.Assoc.,2014,57,7,614-623 [Non-Patent Document 3] Biol.Ther., 2012.2.3.1-22 [Non-Patent Document 4] Drug Design Development Therapy,2013,3,7,711-722 [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention has been made to solve the aforementioned problems and provides a method for producing an IgG Fc fusion protein to increase protein expression levels.

[0008] Another object of the present invention is to provide a method for producing a target protein, which includes culturing cells that produce the target protein using the production method described above.

[0009] Another object of the present invention is to provide a pharmaceutical composition comprising a therapeutic protein and a pharmaceutically acceptable carrier produced by the manufacturing method described above.

[0010] However, the technical problems that this invention aims to solve are not limited to those described above, and other problems not mentioned can be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0011] To solve the above problems, the present invention has confirmed that optimizing the culture conditions of cells producing fusion proteins having an IgG Fc domain improves the productivity and quality of the fusion protein. Specifically, by culturing cells at a normal culture temperature (35.0°C to 38.0°C) for a certain period, and then reducing the culture temperature to 28.0°C to 35.0°C, an increase in the productivity of the fusion protein and suppression of the formation of aggregates of the fusion protein were confirmed, and the present invention was completed based on this.

[0012] In order to achieve the above object, the present invention provides a method for producing a protein in which a soluble extracellular domain of a vascular endothelial growth factor (VEGF) receptor and a human immunoglobulin G (IgG) Fc domain are fused in cell culture, and the method comprises culturing cells at a reduced temperature of 28.0°C to less than 35.0°C in order to increase the expression level of the fusion protein.

[0013] According to a preferred embodiment of the present invention, the fusion protein produced by the above method may have reduced aggregates.

[0014] According to a preferred embodiment of the present invention, the cell culture may be a large-scale cell culture.

[0015] According to another preferred embodiment of the present invention, the cell culture may be any one selected from the group consisting of a batch culture method, a repeated batch culture method, a fed-batch culture method, a repeated fed-batch culture method, a continuous culture method, and a perfusion culture method.

[0016] According to still another preferred embodiment of the present invention, the cell culture may be a fed-batch cell culture.

[0017] According to another preferred embodiment of the present invention, the cells may be mammalian cells.

[0018] According to still another preferred embodiment of the present invention, the mammalian cells may be CHO cells.

[0019] According to another preferred embodiment of the present invention, the CHO cells may be any one cell line selected from the group consisting of DG44, DXB-11, K-1, and CHO-S.

[0020] According to yet another preferred embodiment of the present invention, the culture temperature from the start date of culturing to the temperature change may include a temperature range of 33.0°C to less than 38.0°C.

[0021] According to yet another preferred embodiment of the present invention, the reduced temperature may be 30.0°C to 34.0°C.

[0022] According to another preferred embodiment of the present invention, the culturing period from the start date of culturing to the temperature change may be 1 day to 5 days.

[0023] According to yet another preferred embodiment of the present invention, the culturing period after the temperature reduction may be 2 days to 15 days.

[0024] According to another preferred embodiment of the present invention, the total of the culturing period before the temperature change and the culturing period after the temperature change may be 3 days or more.

[0025] According to another preferred embodiment of the present invention, the soluble extracellular domain of the VEGF receptor may include the immunoglobulin-like domain 2 of the first VEGF receptor and the immunoglobulin-like domain 3 of the second VEGF receptor.

[0026] According to yet another preferred embodiment of the present invention, the produced protein may be a therapeutic protein.

[0027] The present invention also provides a method for producing a target protein, which includes culturing cells that produce the target protein by the above-described production method.

[0028] According to a preferred embodiment of the present invention, it may further include a step of recovering the target protein from the culture solution in which the cells that produce the target protein are cultured.

[0029] According to yet another preferred embodiment of the present invention, the target protein may be a therapeutic protein.

[0030] Furthermore, the present invention provides a pharmaceutical composition comprising a therapeutic protein produced by the manufacturing method described above and a pharmaceutically acceptable carrier.

[0031] However, the technical problems that the present invention aims to solve are not limited to those described above, and other problems not mentioned can be clearly understood by those skilled in the art from the following description. [Effects of the Invention]

[0032] The present invention increases cell growth and viability, increases the expression level of the fusion protein, and suppresses aggregate formation by adding a step of culturing cells that produce a fusion protein having an IgG Fc domain at a reduced culture temperature, thereby increasing the productivity and improving the quality of the fusion protein, and enabling the mass production and supply of the fusion protein. [Brief explanation of the drawing]

[0033] [Figure 1] This graph analyzes the changes in cell growth and cell viability of aflibercept-producing cells depending on the culture temperature. [Figure 2] This graph shows the combined number of aflibercept-producing cells normalized by IVCs (Y-axis; IVCs [normalized 10⁹ cells × days / L]) over time (X-axis; culture time [days]). [Figure 3] This graph shows how the specific production rate of aflibercept-producing cells changes with different culture temperatures. [Figure 4] This graph shows the changes in aflibercept expression levels depending on the culture temperature of cells, as analyzed by HPLC (high-performance liquid chromatography). [Figure 5] This graph analyzes the changes in cell growth and cell viability of aflibercept-producing cells at low culture temperatures. [Figure 6]This graph shows the changes in aflibercept expression levels in cells that produce aflibercept, analyzed using HPLC (high-performance liquid chromatography) at low culture temperatures. [Figure 7] This graph shows the changes in cell growth and cell viability under different culture temperatures for cells producing aflibercept in a 2L bioreactor. [Figure 8] This graph shows the combined number of viable cells (Y-axis; IVC [normalized 10⁹ cells × days / L]) normalized by IVC, for aflibercept-producing cells over time (X-axis; culture time [days]) in a 2L bioreactor. [Figure 9] This graph shows how the specific production rate of aflibercept-producing cells changes with different culture temperatures in a 2L bioreactor. [Figure 10] This graph shows the changes in aflibercept expression levels in cells producing aflibercept in a 2L bioreactor, analyzed using protein A-HPLC (high-performance liquid chromatography). [Figure 11] This graph shows the changes in aggregated proteins in cells producing aflibercept in a 2L bioreactor, analyzed by SE-HPLC (size exclusion high-performance liquid chromatography). [Modes for carrying out the invention]

[0034] The present invention will be described in more detail below. As mentioned above, with the increasing demand for recombinant therapeutic proteins, much research has been conducted on improving cell growth, viability, and protein production and quality through improvements in cell selection, culture medium optimization, and control of the culture process. In relation to protein production, there is a need for methods that can increase protein production or suppress the generation of misfolded / aggregated proteins, as well as the generation of deamided proteins and amino acid substitutions / deletions, thereby preventing safety issues such as immunogenicity and the complexity of the purification process in animal cell culture.

[0035] As a result, the inventors confirmed that culturing cells at a normal culture temperature (35.0°C to 38.0°C) for a certain period, followed by culturing at a reduced culture temperature of 28.0°C to 35.0°C, increases the productivity of fusion proteins having an IgG (Immunoglobulin G) Fc domain and suppresses the formation of aggregates of the fusion protein. By providing a method for producing fusion proteins having an IgG Fc domain in which the protein expression level is increased in cell culture, the inventors have found a solution to the aforementioned problems. The method for producing fusion proteins having an IgG Fc domain of the present invention increases cell growth and cell viability, increases the expression level of the fusion protein, suppresses the formation of aggregates, and consequently increases the productivity and improves the quality of the fusion protein, thereby enabling the mass production and supply of the fusion protein.

[0036] All technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which the present invention pertains.

[0037] In this invention, "fusion protein having an IgG Fc domain" means a protein bound to the Fc region, which is the invariant region of human immunoglobulin G (IgG). In this case, "protein" in this invention means a polymer of several or more amino acids connected by peptide bonds.

[0038] In the present invention, the "amino acid polymer" may be human VEGF receptors 1 and 2, and preferably, the extracellular domains of VEGF receptors 1 and 2 may be used.

[0039] In the present invention, the "Fc region" is an invariant region of the antibody, and human IgG1, IgG2, IgG3, and IgG4 may be used, preferably the Fc region of IgG1.

[0040] The present invention provides a method for producing a protein in which the soluble extracellular domain of a vascular endothelial growth factor (VEGF) receptor and the human immunoglobulin G (Immunoglobulin G) Fc domain are fused in cell culture, wherein the method involves culturing the cells at a reduced temperature of 28.0°C to less than 35.0°C in order to increase the expression level of the fusion protein.

[0041] In the method for producing the fusion protein, the cell culture may be a large-scale cell culture, and the cell culture method may be any commonly used cell culture method. For example, the cell culture method may be one or more selected from the group consisting of batch culture, repeated batch culture, fed-batch culture, repeated fed-batch culture, continuous culture, and perfusion culture, although this is not limited to the above.

[0042] The aforementioned "batch culture method" is a culture method in which a small amount of seed culture medium is added to the culture medium, and cells are grown without adding new medium or draining the culture medium during culture. The aforementioned "continuous culture method" is a culture method in which medium is continuously added and continuously drained during culture. Perfusion culture is also included in the continuous culture method. The aforementioned "fed-batch culture method" is intermediate between the batch culture method and the continuous culture method, and is therefore also called the semi-batch culture method. Medium is added continuously or sequentially during culture, and although continuous draining of the culture medium is performed as in the continuous culture method, cells are not washed away. In the present invention, any of the above culture methods may be used, but preferably the fed-batch culture method or the continuous culture method may be used, and particularly preferably the fed-batch culture method may be used.

[0043] In the present invention, the cells used for the expression of the Fc fusion protein can be any stable cell line capable of sustained expression of the fusion protein, and are preferably mammalian cells. More preferably, commonly used animal cultured cells such as CHO cells, HEK cells, COS cells, 3T3 cells, myeloma cells, BHK cells, HeLa cells, and Vero cells are used, and CHO cells are particularly preferred when the goal is high-volume expression. Furthermore, in order to produce the desired protein, it is especially preferable to use cells suitable for introducing the desired gene, such as dhfr-CHO cells (Proc. Natl. Acad. Sci. USA, 1980, 77, 4216-4220), which are CHO cells lacking the DHFR gene, or CHO K-1 cells (Proc. Natl. Acad. Sci. USA, 1968, 60, 1275). The CHO cells are preferably DG44, DXB-11, K-1, or CHO-S strains, with K-1 strain being particularly preferred. The vector can be introduced into the host cells by methods such as calcium phosphate, DEAE dextran, electroporation, or lipofection.

[0044] In the method for producing a protein in which the soluble extracellular domain of the VEGF receptor and the human IgG Fc domain are fused according to the present invention, the culture temperature from the start of culture until the temperature change can be selected and used according to the culture temperature normally used depending on the cell type. For example, the temperature range normally used to culture mammalian cells may be 33.0°C to less than 38.0°C, and is particularly preferably 37.0°C. In one preferred embodiment of the present invention, cells overexpressing recombinant aflibercept were cultured at 37.0°C from the start of culture until the temperature change to grow the cells.

[0045] In this invention, the timing of temperature changes is determined by the expression level of the target protein. Specifically, the optimal timing for temperature changes can be determined by conducting the experiment shown in Example 3, but since the final cell density differs depending on the cells used and the culture conditions, generally, 1 × 10⁻⁶ 6 cells / mL~1×10 8A cell / mL ratio is preferable.

[0046] The present invention relates to a method for increasing cell-level productivity and suppressing aggregate formation when culturing CHO cells into which a protein-coding gene has been introduced for the purpose of producing the aforementioned fusion protein, characterized in that the cells are cultured at a normal culture temperature from 1 day to 5 days after the start of culture, and then the culture temperature is reduced. The period from the temperature change to the end of culture may generally be 1 to 30 days, and preferably 2 to 15 days. The total of the culture period before and after the temperature change may be 3 days or more. Specifically, in a method for culturing cells that produce the aforementioned fusion protein to produce the protein, the cells are cultured at a normal culture temperature for a certain period, and then subsequently cultured at a reduced temperature. Here, the normal culture temperature is generally 33.0°C to 38.0°C, which is a temperature suitable for cell proliferation of cells derived from homeothermic animals, and 37.0°C is the most common.

[0047] In the present invention's method for producing a protein in which the soluble extracellular domain of the VEGF receptor is fused with the human IgG Fc domain, the reduced culture temperature refers to a temperature range lower than the normal culture temperature, and the optimal reduced culture temperature is determined by the expression level of the target protein. Therefore, in this invention, as described in Example 2, experiments were conducted to determine the optimal reduced culture temperature range in which the target protein is maximally expressed. While the optimal reduced temperature can be determined through experiments like those in Example 2, the final cell density differs depending on the type of cells used and the culture conditions. Therefore, the optimal reduced culture temperature may preferably be 28.0°C to 35.0°C, and more preferably 30.0°C to 34.0°C.

[0048] In the method for producing a protein in which the soluble extracellular domain of a VEGF receptor is fused with a human IgG Fc domain of the present invention, the soluble extracellular domain of the VEGF receptor may include the immunoglobulin-like domain 2 of the first VEGF receptor and the immunoglobulin-like domain 3 of the second VEGF receptor. Specifically, the protein produced by the production method of the present invention may be a therapeutic protein.

[0049] In a preferred embodiment of the present invention, cells overexpressing recombinant aflibercept are cultured at a temperature of 37.0°C from the start of culture until the temperature is changed, and the cell density in the flask is 8 × 10⁶. 6 The cells were cultured for 3 days until the cell / mL concentration was reached. Then, the temperature was reduced to 32.0°C and fed-batch culture was performed according to the supply schedule in Table 1.

[0050] As shown in Figure 1, cells grown in flasks at temperatures lower than normal culture temperatures showed even higher viability than cells subsequently cultured at 37.0°C (control group), resulting in an increased culture period and, consequently, an increase in protein expression, as shown in Figure 4. On the other hand, as shown in Figures 2 and 3, cells grown in flasks at temperatures lower than normal culture temperatures did not affect the number of IVCs or the total amount of expressed protein (PV = culture volume × expression level), and therefore did not show an increase in the specific production rate.

[0051] In another preferred embodiment of the present invention, cells overexpressing recombinant aflibercept are cultured in a flask at a culture temperature of 37.0°C from the start of culture until the temperature change, with a cell density of 8 × 10⁶. 6 The cells were cultured for 2 days until the cell / mL concentration was reached. Then, the temperature was reduced to 30.0°C, 32.0°C, or 34.0°C, and fed-batch culture was performed according to the supply schedule in Table 1.

[0052] As shown in Figure 5, cell concentration increased when cultured at a reduced temperature of 34.0°C in a flask compared to other temperatures, but as shown in Figure 6, the greatest increase in protein expression was observed when cultured at a reduced temperature of 32.0°C.

[0053] In another preferred embodiment of the present invention, cells overexpressing recombinant aflibercept are cultured in a bioreactor at a culture temperature of 37.0°C from the start of culture until the temperature change, with a cell density of 4 × 10⁶. 6 Cells / mL or 8 × 10 6 The cells were cultured for one or two days until the cell / mL concentration was reached. Then, the temperature was reduced to 32.0°C and fed-batch culture was performed according to the supply schedule in Table 1. The pH of the culture medium in the bioreactor varied depending on the cells being cultured, but was generally 6.8 to 7.6, preferably 6.8 to 7.4. Furthermore, the dissolved oxygen (DO) in the culture medium in the bioreactor was generally 20% to 60%, preferably 30% to 50%, and more preferably 40%.

[0054] Unlike flask culture (Erlenmeyer flask), a bioreactor is used to cultivate 8 × 10⁶ cells at a normal culture temperature (37.0°C). 6 Cells cultured to a cell density of cells / mL, and then cultured at a temperature lower than the normal culture temperature, did not show an effect on the number of IVCs, as shown in Figure 8. However, as shown in Figure 9, the total amount of expressed protein (PV = culture volume × expression level) increased, leading to an increase in the specific production rate. Furthermore, as shown in Figure 7, the culture period increased, resulting in an overall increase in protein expression, as shown in Figure 10. In addition, as shown in Figure 11, it was confirmed that fewer aggregates were formed and monomer purity increased.

[0055] Therefore, the method for producing a fusion protein having an IgG Fc domain according to the present invention can increase the productivity of the fusion protein by increasing the growth and viability of cells producing the fusion protein having an IgG Fc domain through optimization of cell culture conditions.

[0056] Furthermore, the method for producing a fusion protein having an IgG Fc domain according to the present invention can improve the method of culturing mammalian cells, increase the productivity of the fusion protein, and suppress the formation of protein aggregates that affect the quality of the fusion protein, thereby providing a fusion protein of improved quality.

[0057] The method of the present invention is characterized by increasing the productivity of the desired protein and suppressing the formation of aggregate components when culturing cells that produce the desired protein to produce the fusion protein. Therefore, it is useful in improving the purification process by suppressing the productivity and formation of aggregate components of aflibercept, a protein in which the ligand-binding portion of the anti-VEGF receptor is fused to the Fc region of IgG1.

[0058] Furthermore, the present invention provides a method for producing a target protein, which includes culturing cells that produce the target protein using the production method described above.

[0059] The method for producing the target protein of the present invention may further include a step of recovering the target protein from a culture medium in which cells that produce the target protein are cultured.

[0060] The target protein produced by the method for producing the target protein described above may also be a therapeutic protein, and the produced therapeutic protein may be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier.

[0061] The pharmaceutical composition of the present invention may be manufactured using pharmaceutically acceptable and physiologically tolerable adjuvants in addition to the active ingredient, and such adjuvants may include excipients, disintegrants, sweeteners, binders, coatings, leavening agents, lubricants, lubricants, or solubilizers such as flavoring agents. The pharmaceutical composition of the present invention can be suitably formulated as a pharmaceutical composition containing, in addition to the active ingredient, one or more pharmaceutically acceptable carriers for administration. Acceptable pharmaceutically acceptable carriers in compositions formulated as liquid solutions are sterile and biocompatible, and can be saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components, and other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants can be added to formulate the composition into injectable dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets.

[0062] The pharmaceutical compositions of the present invention can be administered to mammals, including humans, via a variety of routes, depending on the type of therapeutic protein produced. For example, they can be administered orally or parenterally, and parenteral administration methods, though not limited to these, may include intravenous, intramuscular, intra-arterial, intra-bone marrow, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, rectal, or intravitreal injection.

[0063] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, medical condition, diet, administration time, route of administration, excretion rate, and response sensitivity, and a typical, experienced physician can easily determine and prescribe a dosage that is effective for the desired treatment or prevention.

[0064] The present invention will be described in more detail below through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples. [Examples]

[0065] [Manufacturing example] Production of recombinant aflibercept expression vectors and expressing cell lines Recombinant aflibercept was cloned as a fusion protein using an improved vector obtained by removing shGH, His tag, and TEV site from pSGHV0 (GenBank Accession No. AF285183), and its extracellular secretion was achieved using a protein-specific signal sequence.

[0066] Furthermore, the GS system was introduced as a selection marker to construct a stable cell line that sustainably expresses the aforementioned fusion protein, and for this purpose, the mouse glutamine synthase gene was inserted into the vector. A Kozac sequence was further inserted into the signal sequence to increase the expression level. Next, the clone thus produced was introduced into the CHO-K1 cell line (ATCC, Cat.CCL-61) to further select for methionine sulfoximine (MSX) and secure a stable cell line. However, those with ordinary knowledge in this field can appropriately select and apply commonly used vectors and cell lines depending on the given circumstances.

[0067] [Example 1] Confirmation of the effects of reduced temperature culture on cell state and IgG Fc fusion protein production. Cells overexpressing recombinant aflibercept, produced in the above manufacturing example, were inoculated in two 125 mL Erlenmeyer flasks into a culture medium supplemented with plant-derived hydrolyzed protein at the same concentration and conditions, and cultured in a CO2 incubator with shaking at 37.0°C. After growing the cells in batch cultures, the cell concentration reached approximately 8 × 10⁶. 6When the feed rate was cell / mL, the temperature was reduced to 32.0°C and the cells were cultured in a fed-batch state. The feed schedule for the experimental conditions is summarized in Table 1 below. The volume of the fed-batch feed is expressed as a percentage of the initial culture volume in the bioreactor. Cell samples were taken daily from the culture to measure the levels of viable cells, cell viability, expression levels, and specific production rates. "Integral viable cell count" or "IVC (Integral viable cell)" refers to the average density of viable cells over the course of the culture, multiplied by the time the culture is performed. When the amount of protein produced is proportional to the number of viable cells present over the course of the culture, the density of integrated viable cells was used to estimate the amount of protein produced over the course of the culture. The integrated viable cell count (IVC) was calculated by measuring using a cell density test performed by staining with trypan blue and measuring under a microscope, and then normalized by the IVC. The IVC was determined by calculating the arithmetic mean of the harvest-day integrated viable cell density for all experimental conditions tested. Expression levels were measured by protein A-HPLC to calculate specific production rates.

[0068] [Table 1]

[0069] As a result, cells cultured at low temperatures in 125 mL Erlenmeyer flasks did not show an increase in specific production rates, as the number of IVCs and the total amount of expressed protein (PV = culture volume × expression level) were not affected (Figures 2 and 3). However, cells grown at low temperatures developed even higher cell viability (Figure 1), leading to increased culture duration and expression levels (Figure 4).

[0070] [Example 2] Confirmation of the optimal temperature for changing the expression level of IgG Fc fusion protein The cells overexpressing recombinant aflibercept produced in the above production example were inoculated into a medium supplemented with a plant-derived hydrolyzed protein in three 125 mL Erlenmeyer flasks at the same concentration and conditions, and cultured with shaking in a CO2 incubator at 37.0 °C. After growing the cells in batch culture, when the cell concentration reached about 8×10 6 cells / mL, the temperature was reduced to 30.0 °C, 32.0 °C, or 34.0 °C, respectively, for fed-batch culture. The supply schedule for the experimental conditions was carried out as shown in Table 1 of Example 1. Various states of the cells were measured as described in Example 1.

[0071] As a result, the cells cultured at a low temperature of 34.0 °C had the most significant increase in cell concentration without a change in viability (Figure 5). However, the expression level of the cells cultured at a low temperature of 32.0 °C increased the most compared to other temperatures (Figure 6).

[0072] [Example 3] Bioreactor verification experiment The cells overexpressing recombinant aflibercept produced in the above production example were inoculated into a plant-derived feed medium in a New Brunswick Scientific (NBS) bioreactor and stirred at a pH of 6.8 - 7.4 and a speed of 80 rpm. When the cell concentration in the bioreactor reached 4×10 6 cells / mL or 8×l0 6 cells / mL on the first or second day, the temperature was changed from 37.0 °C to 32.0 °C for culture. The supply schedule for the experimental conditions was carried out as shown in Table 1 of Example 1. Various states of the cells were measured as described in Example 1.

[0073] As a result, different from the 125 mL Erlenmeyer flask culture, when the cell concentration reached 8×10 6Cells cultured at low temperatures at cell / mL did not show an effect on the number of IVCs, but the total amount of expressed protein (PV = culture volume × expression level) increased, leading to an increase in specific production rates, and the expression level increased with increasing culture period (Figures 7-10). Samples cultured under the above temperature conditions were separated for protein using a protein A column and analyzed by SE-HPLC. From the results of the SE-HPLC analysis, the monomer ratio was calculated, and the ratio of aggregates (high molecular weight impurities) to fragments (low molecular weight impurities) was calculated. The experimental results confirmed that fewer aggregates were formed and monomer purity increased in cultures with reduced temperature (Figure 11).

[0074] The above results demonstrate that, through the method for producing a protein in which the soluble extracellular domain of the VEGF receptor and the human IgG Fc domain are fused according to the present invention, aflibercept, represented by the fusion protein, can be produced with high quality while suppressing aggregate formation. Furthermore, its expression level can be significantly increased through culture at reduced temperatures, thereby increasing productivity.

[0075] In the production of fusion proteins using cell culture, the culture conditions that simultaneously satisfy both protein quality and productivity vary depending on the type of protein, and numerous trials and errors are necessary to find the optimized conditions. As demonstrated in the above examples, the method for producing fusion proteins of the present invention is optimized for the production of proteins in which the soluble extracellular domain of the VEGF receptor and the human IgG Fc domain are fused (e.g., aflibercept), and the productivity and quality of the protein can change significantly when the type of protein changes. Therefore, when the type of target protein to be produced changes, it is essential to readjust the optimized conditions to improve quality and productivity and to verify them.

[0076] The above description of the present invention is illustrative, and those skilled in the art will understand that the invention can be easily modified in other specific forms without altering the technical idea or essential features. Therefore, the embodiments described above should be understood to be illustrative and not limiting in all respects. [Industrial applicability]

[0077] The method for producing a protein in which the soluble extracellular domain of a VEGF receptor and the human IgG Fc domain are fused, as provided in the present invention, increases cell growth and cell viability by adding a step of culturing the cells that produce the fusion protein at a reduced culture temperature, thereby increasing the productivity of the fusion protein, suppressing aggregate formation of the fusion protein, and improving quality, thereby enabling the mass production and supply of the fusion protein.

[0078] Furthermore, the protein produced by the production method of the present invention is a therapeutic protein that can provide pharmaceutical compositions in a form suitable for therapeutic purposes, and thus has great industrial applicability.

Claims

1. A method for producing aflibercept in cell culture, a) Culturing cells capable of producing aflibercept in a temperature range of 33°C to 38°C; and b) To increase the expression level of aflibercept, the cell density should be 1 × 10⁻⁶ ６ cells / ml ~ 1 x 10 ８ When the cell / ml is reached, the cells are cultured at a reduced temperature of 30°C to 35°C. A method comprising the above, wherein the reduced temperature in b) is lower than the temperature selected in a), and the aflibercept produced by the method is aflibercept in which the amount of aggregates is reduced compared to aflibercept produced by culturing the cells without using the temperature range in b) which is lower than the temperature at which the cells are cultured in a).

2. The method according to claim 1, wherein the cell culture is a large-scale cell culture.

3. The method according to claim 2, wherein the cell culture is one or more selected from the group consisting of batch culture, repeated batch culture, fed-batch culture, repeated fed-batch culture, continuous culture, and perfusion culture.

4. The method according to claim 3, wherein the cell culture is fed-batch cell culture.

5. The method according to claim 1, wherein the cells are mammalian cells.

6. The method according to claim 5, wherein the mammalian cell is a CHO cell.

7. The method according to claim 6, wherein the CHO cells are one cell line selected from the group consisting of DG44, DXB-11, K-1, and CHO-S.

8. The method according to claim 1, wherein the culture period of (a) is 1 to 5 days.

9. The method according to claim 1, wherein the culture period for (b) is 2 to 15 days.

10. The method according to claim 1, wherein the total of the culture periods in a) and b) is 3 days or more.

11. A method for producing aflibercept, the method comprising culturing cells that produce aflibercept by the method according to any one of claims 1 to 10.

12. The method according to claim 11, further comprising the step of recovering the aflibercept from a culture medium in which the cells producing the aflibercept are cultured.

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