WO2017065559A1 - Method for producing fusion protein having igg fc domain - Google Patents

Abstract

The present invention relates to a method for preparing a fusion protein having an IgG Fc domain and, specifically, to a method for preparing a fusion protein having an IgG Fc domain, the method additionally comprising a step of culturing cells, which produce the fusion protein, at a decreased culture temperature, thereby increasing cell growth and cell viability so as to increase fusion protein productivity and inhibiting aggregate generation so as to improve quality and production yield.

Description

Method for producing a fusion protein having an IGG FC domain

This application claims the priority of Korean Patent Application No. 10-2015-0144330, filed October 15, 2015 and Korean Patent Application No. 10-2016-0132633, filed October 13, 2016. It is a reference of the present application.

The present invention relates to a fusion protein having a human immunoglobulin G (IgG) Fc domain (particularly, a fusion of extracellular domain of human vascular endothelial growth factor (VEGF) receptor and human immunoglobulin G (IgG) Fc domain). The present invention relates to a method for producing a protein (eg, Aflibercept).

Vascular Endothelial Growth Factor (VEGF) is an important factor for increasing angiogenesis and vascular permeability. In particular, VEGF is overexpressed in tumor cells and abnormally promotes angiogenesis and tumor proliferation (Oncogene, 2004, 23, 1745-1753). In addition, abnormal angiogenesis has been reported to be importantly related to other diseases in addition to tumor development. Abnormal angiogenesis in the mechanism through VEGF is associated with ocular diseases such as wet macular degeneration, diabetic retinopathy, and retinal vein obstructive macular edema (J. Korean Med. Assoc., 2014 ,, 57,7, 614-623 ).

Pegaptanib (RNA aptamer), Ranibizumab (monoclonal IgG antibody fragment (Fab)), Bevacizumab (monoclonal IgG antibody) are used as therapeutic agents for such ophthalmic diseases. , Aflibercept (VEGFR1 and VEGFR2 fused with IgG1 Fc) was approved in the United States in 2011 for the treatment of wet macular degeneration (Biol. Ther., 2012, 2, 3, 1-22; Drug Design Development Therapy , 2013, 3, 7, 711-722).

Due to the increasing demand for such therapeutic recombinant proteins, many efforts have been made to improve cell growth, viability and protein production and quality through improved cell selection, media optimization and culture process control. Many proteins and polypeptides produced by cell culture are produced by producing and isolating cells by batch or fed-batch methods at a constant temperature or pH for a period of time. Therefore, yield and quality can be influenced by cell culture conditions. Regulation and optimization of cell culture conditions related to protein production in animal cell cultures to increase protein production, inhibit the formation of misfolded / aggregated proteins, inhibit the production of protein deamidates or amino acid substitutions and deletions There is a need for a method that can prevent safety problems such as and the like, and the complexity of the purification process.

The present invention has been made to solve the above problems, and provides a method for producing an IgG Fc fusion protein for increasing the amount of protein expression.

Another object of the present invention is to provide a method for producing a target protein comprising culturing a cell producing the target protein by the above-described production method.

It is still another object of the present invention to provide a pharmaceutical composition comprising a therapeutic protein prepared by the above-described preparation method and a pharmaceutically acceptable carrier.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to solve the above problems, the present invention was confirmed that the productivity and quality of the fusion protein is improved by optimizing the culture conditions of the cells producing the fusion protein having the IgG Fc domain. Specifically, after culturing at a normal culture temperature (35.0 ℃ ~ 38.0 ℃) for a period of time, the culture temperature is reduced to 28.0 ℃ ~ 35.0 ℃ by culturing, thereby increasing the productivity of the fusion protein and aggregates (aggregates) of the fusion protein ) Inhibition of production was confirmed and based on this, the present invention was completed.

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

According to a preferred embodiment of the present invention, the fusion protein prepared by the above method may be one in which aggregates are reduced.

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

According to another preferred embodiment of the present invention, the cell culture is a batch culture (batch culture), repeated batch culture (repeated batch culture), fed-batch culture, repeated fed-batch culture (repeated fed-batch culture) It may be any one selected from the group consisting of, continuous culture (peruous culture) and perfusion culture (perfusion culture).

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

According to another preferred embodiment of the present invention, the cell may be a mammalian cell.

According to another preferred embodiment of the present invention, the mammalian cell may be a CHO cell.

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.

According to another preferred embodiment of the present invention, the culture temperature before the temperature change from the start of the culture may include a temperature range of less than 33.0 ℃ to 38.0 ℃.

According to another preferred embodiment of the present invention, the reduced temperature may be 30.0 ℃ to 34.0 ℃.

According to another preferred embodiment of the present invention, the incubation period may be from 1 to 5 days before the temperature change from the start of the culture.

According to another preferred embodiment of the present invention, the incubation period after the temperature decrease may be 2 to 15 days.

According to another preferred embodiment of the present invention, the sum of the culture period before the temperature change and the culture period after the temperature change may be 3 days or more.

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

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

The present invention also provides a method for producing a target protein comprising culturing a cell producing the target protein by the above-described production method.

According to a preferred embodiment of the present invention, the cell producing the target protein may further comprise the step of recovering the target protein from the culture medium.

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

The present invention also provides a pharmaceutical composition comprising a therapeutic protein prepared by the above-described preparation method and a pharmaceutically acceptable carrier.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention further comprises the step of culturing cells producing a fusion protein having an IgG Fc domain at a reduced culture temperature, thereby increasing cell growth and cell viability, increasing the expression level of the fusion protein, and aggregates. Inhibiting production, consequently increasing the productivity and improving the quality of the fusion protein, allowing mass production and supply of the fusion protein.

1 is a graph analyzing the change of cell growth and cell viability according to the culture temperature of cells producing Aflibercept (Aflibercept).

FIG. 2 shows the integrated viable cell count (Y axis; IVC [normalized 10 ⁹ cells × day) normalized to IVC of cells producing Aflibercept over time (X axis; incubation time [day]). / L]).

3 is a graph showing a specific production rate change with the culture temperature of the cells producing Aflibercept (Aflibercept).

Figure 4 is a graph of the change in the expression amount according to the culture temperature of the cells producing Aflibercept (HPLC) by high performance liquid chromatography.

5 is a graph analyzing the change of cell growth and cell viability according to the cold culture temperature of the cells producing Aflibercept (Aflibercept).

FIG. 6 is a graph of HPLC (high performance liquid chromatography) analysis of changes in expression level according to cold culture temperature of cells producing Aflibercept.

FIG. 7 is a graph illustrating changes in cell growth and cell viability according to culture temperature of cells producing Aflibercept in a 2L bioreactor.

FIG. 8 shows the integrated viable cell count (Y axis; IVC [normalized to IVC) of cells producing Aflibercept over time (X axis; incubation time [day]) in a 2L bioreactor. Normalized 10 ⁹ cells × day / L]).

FIG. 9 is a graph showing changes in specific production rate according to the culture temperature of cells producing Aflibercept in a 2L bioreactor.

FIG. 10 is a graph of protein A-HPLC (High Performance Liquid Chromatography) analyzing the change in the expression level according to the culture temperature of cells producing Aflibercept in a 2L bioreactor.

FIG. 11 is a graph of SE-HPLC (size exclusion high performance liquid chromatography) analysis of changes in aggregated protein according to the culture temperature of cells producing Aflibercept in a 2L bioreactor.

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

As described above, due to the increasing demand for therapeutic recombinant proteins, many efforts have been made to improve cell growth, viability and protein production and quality through improved cell selection, media optimization and culture process control. Safety issues such as immunogenicity by inhibiting the increase of protein production or the production of misfolded / aggregated proteins, the inhibition of protein deamidation or amino acid substitution and deletion through regulation and optimization of cell culture conditions related to protein production There is a need for a method that can prevent the complexity of the refining process.

Accordingly, the present inventors incubated at a normal culture temperature (35.0 ° C. to 38.0 ° C.) for a certain period of time, and then cultured by reducing the culture temperature to 28.0 ° C. to 35.0 ° C., thereby producing a fusion protein having an IgG (Immunoglobulin G) Fc domain. It was confirmed that this increase and the production of aggregates (aggregates) of the fusion protein is suppressed, and the solution of the above-described problem was sought by providing a method for producing a fusion protein having an IgG Fc domain, the protein expression amount is increased in cell culture. The method for producing a fusion protein having an IgG Fc domain of the present invention increases the cell growth and cell viability by performing a step of culturing the cells producing the fusion protein having an IgG Fc domain at a reduced culture temperature, thereby increasing the cell growth and cell viability. Increasing the amount of expression, inhibiting the production of aggregates (aggregates), as a result of increasing the productivity and quality of the fusion protein, the mass production and supply of the fusion protein is possible.

All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In the present invention, "fusion protein having an IgG Fc domain" means a protein bound to an Fc region which is a constant region of human immunoglobulin G (IgG). At this time, in the present invention, "protein" means a peptide bond of several or more amino acids.

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

In the present invention, "Fc region" may be used as a constant region of the antibody human IgG1, IgG2, IgG3 and IgG4, preferably the Fc region of IgG1 can be used.

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 Fc domain are fused in cell culture. Provided are methods for culturing cells at reduced temperatures below 28.0 ° C. to less than 35.0 ° C. to increase the amount.

In the production method of the fusion protein, the cell culture may be a large-scale cell culture, the cell culture method may use a conventionally used cell culture method. For example, the cell culture method is not limited to this, but batch culture, repeated batch culture, fed-batch culture, repeated fed-batch culture , At least one selected from the group consisting of continuous culture and perfusion culture.

The "batch culture method" is a culture method in which a small amount of seed culture solution is added to the medium, and the cells are grown without adding a medium or releasing the culture medium during the culture. The " continuous culture method " is a culture method in which a medium is continuously added during culturing and also continuously discharged. In addition, the continuous culture method also includes perfusion culture. Since the "fed-value cultivation method" is halfway between the batch cultivation method and the continuous culturing method, it is also called a semi-batch culture, and the medium is added continuously or sequentially during the cultivation. It is a culture method in which culture medium is discharged but cells are not leaked. In the present invention, any of the above culturing methods may be used. Preferably, a fed-batch culture method or a continuous culture method may be used, and particularly preferably, a fed-batch culture method may be used.

Cells used for the expression of the Fc fusion protein in the present invention can be used without limitation as long as it is a stable cell line capable of continuously expressing the fusion protein, preferably may be a mammalian cell. More preferably, animal culture cells commonly used, such as CHO cells, HEK cells, COS cells, 3T3 cells, myeloma cells, BHK cells, HeLa cells, Vero cells, are used. Cells are preferred. In addition, in order to produce a desired protein, in particular, dhfr-CHO cells (Proc. Natl. Acad. Sci. USA, 1980, 77, 4216-4220) or CHO K-1 cells (Proc), which are CHO cells lacking the DHFR gene, are also used. Natl. Acad. Sci. USA, 1968, 60, 1275), and the like, and are preferably cells suitable for introducing a desired gene. Especially as said CHO cells, DG44 strain, DXB-11 strain, K-1 strain or CHO-S strain is preferable, K-1 strain is especially preferable, The introduction of the vector into a host cell is carried out by the calcium phosphate method, DEAE dextran method. , Electroporation, lipofection, or the like can be carried out.

In the method for producing a protein in which the soluble extracellular domain and the human IgG Fc domain of the VEGF receptor of the present invention are fused, the culture temperature from the culture start date until the temperature change can be used by selecting a culture temperature that is commonly used according to the cell type. have. For example, the temperature range typically used for mammalian cell culture may be less than 33.0 ° C to 38.0 ° C, particularly preferably 37.0 ° C. In a preferred embodiment of the present invention, cells overexpressing the recombinant aplibercept were grown at 37.0 ° C. until the temperature change from the start of the culture to grow the cells.

In the present invention, the timing of the temperature change is determined by the expression level of the target protein. Specifically, by performing the experiment shown in Example 3, the optimum temperature change timing can be known, but since the final cell density varies depending on the cells used and the culture conditions, it is generally from 1 × 10 ⁶ cells / ml to Preferred is about 1 × 10 ⁸ cells / ml.

The present invention is a method for suppressing the increase in productivity per cell and aggregates when culturing CHO cells into which a gene encoding a protein is introduced for the purpose of producing the fusion protein. The culture is carried out at a normal culture temperature until 5 days later, and then the culture temperature is reduced. The period from the temperature change to low temperature until the end of the culture is generally 1 day to 30 days, preferably 2 to 15 days. The sum of the incubation period before the temperature change and the incubation period after the temperature change may be 3 days or more. Specifically, the method for producing the protein by culturing the cells producing the fusion protein, characterized in that after culturing at a normal culture temperature for a certain period of time, the culture is continued at a reduced temperature. The typical culture temperature here is generally 33.0 ° C. to 38.0 ° C., which is suitable for cell proliferation of constant temperature animal derived cells, and 37.0 ° C. is most common.

In the method for producing a protein in which the soluble extracellular domain and the human IgG Fc domain of the VEGF receptor of the present invention are fused, a reduced culture temperature means a temperature range lower than a conventional culture temperature, and an optimal reduced culture temperature is a target. It is determined by the expression level of the protein. Thus, in the present invention, experiments were carried out as described in Example 2 to derive an optimal reduced culture temperature range in which the desired protein was maximally expressed. Although the optimal alteration temperature can be known through the same experiment as in Example 2, since the final cell density varies depending on the type of cells used or the culture conditions, the optimal reduced culture temperature is preferably 28.0 ° C to 35.0 ° C, More preferably, it may be 30.0 ° C to 34.0 ° C.

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

In a preferred embodiment of the present invention, the cells overexpressing the recombinant aplybercept is 37.0 ° C. with a culture temperature of 37.0 ° C. from the start of the culture to the temperature change until the cell density reaches 8 × 10 ⁶ cells / mL in the flask. Incubated for Thereafter, the temperature was lowered to 32.0 ° C. and oil culture was carried out according to the feeding schedule of Table 1.

As shown in FIG. 1, the cells growing at a temperature lower than the normal culture temperature in the flask showed higher viability than the cells continuously cultured at 37.0 ° C. (control), thereby increasing the incubation period. As shown, the expression level of the protein was increased. On the other hand, as shown in Figures 2 and 3, the cells growing at a temperature lower than the normal culture temperature in the flask does not affect the IVC number and the total amount of protein expressed (PV = culture volume × expression) because the specific production rate (specific production rate) did not increase.

In another preferred embodiment of the present invention, the cell overexpressing the recombinant aplibercept is 37.0 ° C. until the temperature is changed from the start of the culture until the temperature is changed until the density of the cells in the flask reaches 8 × 10 ⁶ cells / mL. Incubated for days. Thereafter, the temperature was lowered to 30.0 ° C. or 32.0 ° C. or 34.0 ° C., and then cultured with milk according to the feeding schedule of Table 1.

As shown in FIG. 5, the cell concentration is increased compared to other temperatures in the culture at the culture temperature reduced to 34.0 ° C. in the flask, but as shown in FIG. 6, the expression level of the protein in the culture at the culture temperature reduced to 32.0 ° C. is shown. Most increased.

In another preferred embodiment of the present invention, the cell overexpressing the recombinant aplibercept is at a culture temperature of 37.0 ° C. from the start of the culture until the temperature change is 4x10 ⁶ cells / mL or 8x10 ⁶ cells / mL in the bioreactor. Incubate for 1 or 2 days until reaching mL. Thereafter, the temperature was lowered to 32.0 ° C. and oil culture was carried out according to the feeding schedule of Table 1. The pH of the culture medium in the bioreactor is different depending on the cells to be cultured, but may generally be pH 6.8 to 7.6, preferably pH 6.8 to 7.4. In addition, dissolved oxygen (DO) of the culture medium in the bioreactor is generally 20% to 60%, preferably 30% to 50%, more preferably 40% is used.

Unlike the flask culture (Erlenmeyer flask), using a bioreactor incubated to a cell density of 8x10 ⁶ cells / mL at a conventional culture temperature (37.0 ℃) and then changed to a temperature lower than the normal culture temperature cultured cells Although it does not affect the number of IVCs as shown in FIG. 8, as shown in FIG. 9, the total amount of protein expressed (PV = culture volume × expression amount) increased to increase a specific production rate, and FIG. 7. As shown in FIG. 10, the incubation period was increased, and as a result, the protein expression amount was increased as shown in FIG. 10. In addition, as shown in Figure 11 it was confirmed that less aggregates (aggregates) are generated to increase the monomer purity (monomer purity).

Accordingly, the method for producing a fusion protein having an IgG Fc domain of the present invention increases the growth and survival rate of cells producing a fusion protein having an IgG Fc domain through optimization of cell culture conditions, thereby increasing the productivity of the fusion protein. Can be.

In addition, the method for producing a fusion protein having an IgG Fc domain of the present invention improves the method of culturing mammalian cells, thereby increasing the productivity of the fusion protein and inducing the aggregation of protein aggregates that affect the quality of the fusion protein. Production can be inhibited to provide fusion proteins with improved quality.

The method of the present invention is characterized by increasing the productivity of the desired protein and inhibiting the generation of aggregate components when the fusion protein is produced by culturing cells producing the desired protein. Therefore, the ligand-binding portion of the anti-VEGF receptor will help to improve the purification process by inhibiting the production and aggregation of Aflibercept, a protein fused to the Fc region of IgG1.

The present invention also provides a method for producing a target protein comprising culturing a cell producing the target protein by the above-described production method.

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

The target protein prepared by the method for preparing the target protein may be a therapeutic protein, and the prepared therapeutic protein may be provided in a pharmaceutical composition together with a pharmaceutically acceptable carrier.

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant may include excipients, disintegrants, sweeteners, binders, coatings, swelling agents, lubricants, lubricants. Solubilizers, such as an agent or a flavoring agent, can be used. The pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition by containing one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration. Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

The pharmaceutical composition of the present invention can be administered to various mammals, including humans, according to the kind of therapeutic protein prepared. For example, it can be administered orally or parenterally, parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal , Intranasal, intestinal, topical, sublingual, rectal or intravitreal injection administration.

Suitable dosages of the pharmaceutical compositions of the present invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to response of the patient, Usually a skilled practitioner can easily determine and prescribe a dosage effective for the desired treatment or prophylaxis.

Hereinafter, an embodiment of the present invention will be described in more detail. However, these examples are for illustrating the present invention, the scope of the present invention is not limited by these examples.

[Production example]

Preparation of Recombinant Aflibercept Expression Vectors and Expression Cell Lines article

Recombinant Aflibercept was cloned as a fusion protein using an improved vector from which shGH, His tag, and TEV sites were removed from pSGHV0 (GenBank Accession No. AF285183), and a unique signal sequence was used It was allowed to secrete extracellularly.

In addition, the GS system was introduced as a selection marker for constructing a stable cell line expressing the fusion protein. For this purpose, a mouse glutamine synthetase gene was inserted into a vector. Kozac sequence was further inserted into the signal sequence to increase expression. Then, the clone thus prepared was introduced into the CHO-K1 cell line (ATCC, Cat. CCL-61) to proceed with the selection of methionine sulphoximine (MSX) to secure a stable cell line. However, one of ordinary skill in the art may appropriately select and apply vectors and cell lines which are commonly used according to a given situation.

Example 1

Reduced  Cell condition according to temperature culture and IgG Fc  Identification of effects on fusion protein production

Cells overexpressing the recombinant Aflibercept prepared in the above preparation was inoculated in two 125 mL cells in a Erlenmeyer flask at the same concentration and conditions to which the plant-derived hydrolyzed protein was added and 37.0. Shake incubation in a C ₂ CO ₂ incubator. The cells were grown in batch culture, and then fed to a fed-batch culture by lowering the temperature to 32.0 ° C. when the cell concentration was about 8 × 10 ⁶ cells / mL. The feeding schedule for the experimental conditions is summarized in Table 1 below. The feedstock volume is described as a percentage of the starting volume of culture in the bioreactor. Cell samples were taken daily from the culture to determine the level of viable cells, cell viability, expression level and specific production rate. "Integral viable cell" or "IVC (Integral viable cell)" means the average density of viable cells over the course of the culture multiplied by the time the culture is run. When the amount of protein produced is proportional to the number of viable cells present over the course of the culture, integrated viable cell density was used to estimate the amount of protein produced over the course of the culture. Integral viable cells (IVC) were calculated by measuring using a cell density test that was stained with trypan blue and measured microscopically and normalized to IVC. IVC was determined by calculating the arithmetic mean of harvest day integrated viable cell density for all experimental conditions tested. The expression level was measured by Protein A-HPLC and the specific production rate was calculated.

Experimental conditions	37.0 ℃	37.0 → 32.0 ℃
2 days	2.86%	2.86%
3 days	2.86%	2.86%
4 days	2.86%	2.86%
5 days	2.86%	2.86%
6 days	2.86%	2.86%
7 days	2.86%	2.86%
8th	2.86%	2.86%

As a result, the cells cultured in the 125 mL Erlenmeyer flask did not increase the specific production rate because they did not affect the IVC number and the total amount of expressed protein (PV = culture volume x expression). (Figures 2 and 3). However, cells growing at low temperatures resulted in higher cell viability (FIG. 1), thereby increasing culture duration and expression (FIG. 4).

Example 2

Confirmation of Optimal Change Temperature According to the Expression Level of IgG Fc Fusion Protein

Cells overexpressing the recombinant Aflibercept prepared in the above Preparation Example were inoculated in three 125 mL in an Erlenmeyer flask in a medium to which the plant-derived hydrolyzed protein was added at the same concentration and conditions, and 37.0. Shake incubation in a C ₂ CO ₂ incubator. The cells were grown in batch culture, and when the cell concentration was about 8 × 10 ⁶ cells / mL, the cells were fed incubated by lowering the temperature to 30.0 ° C., 32.0 ° C. or 34.0 ° C., respectively. The supply schedule for the experimental conditions was performed as shown in Table 1 of Example 1. Various conditions of the cells were measured as described in Example 1.

As a result, cells cultured at 34.0 ° C. low temperature showed the highest increase in cell concentration without change in viability (FIG. 5). However, the expression level of 32.0 ° C. low temperature cultured cells was most increased compared to other temperatures (FIG. 6).

 Example 3

Bioreactor Identification Experiment

Cells overexpressing the recombinant Aflibercept prepared in the above preparation were used as a plant-derived feed medium in a New Brunswick Scientific (NBS) bioreactor and at a pH of 6.8-7.4 and 80 rpm. Stirred. The bioreactor was incubated by changing the temperature from 37.0 ° C. to 32.0 ° C. when the cell concentration reached ⁴ × 10 ⁶ cell / mL or 8 × 10 ⁶ cell / mL on the first or second day. The supply schedule for the experimental conditions was performed as shown in Table 1 of Example 1. Various conditions of the cells were measured as described in Example 1.

As a result, unlike 125 mL Erlenmeyer flasks, cells cultured at 8 × 10 ⁶ cells / mL at low temperature did not affect the IVC count, but the total amount of protein expressed (PV = culture volume × expression) was increased. The specific production rate was increased and the expression amount increased with the increase of the culture period (Figs. 7 to 10). Samples incubated at the above temperature conditions were separated by Protein A column and analyzed by SE-HPLC. The ratio of monomer was calculated from the result of analysis by SE-HPLC, and the ratio of aggregates as polymer impurities and fragments as low molecular impurities was calculated. As a result, it was confirmed that less aggregates were generated in the culture at reduced temperature, thereby increasing monomer purity (FIG. 11).

The above results can be produced in high quality by inhibiting the generation of aggregates of Aflibercept represented by the fusion protein through the production method of the protein fused with the soluble extracellular domain and the human IgG Fc domain of the VEGF receptor of the present invention. In addition, it is demonstrated that the expression level can be increased significantly through the incubation at reduced temperature, thereby increasing the productivity.

In the production of the fusion protein through cell culture, the culture conditions satisfying the quality and productivity of the protein at the same time is different depending on the type of protein, many trials and errors are required to find the optimized conditions. As demonstrated in the above examples, the fusion protein production method of the present invention is optimized for the production of a protein (eg, aflibercept) fused with a soluble extracellular domain of the VEGF receptor and a human IgG Fc domain. Different levels of protein can significantly alter the productivity and quality of the protein. Therefore, if the type of target protein to be produced is changed, a process of resetting and verifying optimized conditions for improving quality and productivity will be required.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

 The method for producing a protein in which the soluble extracellular domain and the human IgG Fc domain of the VEGF receptor provided by the present invention are fused includes adding a step of culturing the cells producing the fusion protein at a reduced culture temperature, thereby increasing cell growth and cell viability. By increasing the productivity of the fusion protein can be increased and the production of aggregates (aggregates) of the fusion protein can be suppressed to improve the quality, allowing the mass production and supply of the fusion protein.

In addition, the protein produced by the production method of the present invention is a therapeutic protein, which can provide a pharmaceutical composition in a suitable form according to the therapeutic purpose, and thus has great industrial applicability.

Claims (21)

1. 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 Fc domain are fused in cell culture, thereby increasing the expression level of the fusion protein. To incubate the cells at a reduced temperature of from 28.0 ° C. to less than 35.0 ° C.
2. The method of claim 1, wherein the fusion protein produced by the method is reduced in aggregates.
3. The method of claim 1, wherein the cell culture is a large scale cell culture.
4. The method of claim 3, wherein the cell culture is batch culture (batch culture), repeated batch culture (repeated batch culture), fed-batch culture (repeated fed-batch culture), continuous culture (continuous culture) culture) and perfusion culture.
5. The method of claim 4, wherein the cell culture is a fed-batch cell culture.
6. The method of claim 1, wherein the cell is a mammalian cell.
7. The method of claim 6, wherein the mammalian cell is a CHO cell.
8. The method of claim 7, wherein the CHO cells are any one of the cell lines selected from the group consisting of DG44, DXB-11, K-1 and CHO-S.
9. The method of claim 1, wherein the incubation temperature from the incubation date until the temperature change comprises a temperature range of 33.0 ° C. to less than 38.0 ° C. 7.
10. The method of claim 1, wherein the reduced temperature is 30.0 ° C. to 34.0 ° C. 3.
11. The method of claim 1, wherein the incubation period is from 1 to 5 days before the temperature change from the start of the culture.
12. The method of claim 1, wherein the incubation period after temperature reduction is 2 to 15 days.
13. The method of claim 1, wherein the sum of the incubation period before the temperature change and the incubation period after the temperature change is 3 days or more.
14. The method of claim 1, wherein the soluble extracellular domain of the VEGF receptor comprises immunoglobulin-like domain 2 of the first VEGF receptor and immunoglobulin-like domain 3 of the second VEGF receptor.
15. The method of claim 1, wherein the protein produced is a therapeutic protein.
16. A method for producing a target protein comprising culturing a cell producing the target protein by the method of any one of claims 1 to 15.
17. The production method according to claim 16, further comprising the step of recovering the target protein from the culture medium in which the cells producing the target protein are cultured.
18. The method of claim 16, wherein the target protein is a therapeutic protein.
19. A pharmaceutical composition comprising a therapeutic protein prepared by the method of claim 18 and a pharmaceutically acceptable carrier.
20. The method of claim 17, wherein the target protein is a therapeutic protein.
21. A pharmaceutical composition comprising a therapeutic protein prepared by the method of claim 20 and a pharmaceutically acceptable carrier.

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