Methods for producing dual function proteins and derivatives thereof

Abstract

A culture method for producing dual-function proteins with improved pharmacokinetic parameters, high stability, low potential for aggregation to form complexes, and reduced potential for immunogenicity is provided. [Solution] A method for producing a recombinant dual-function protein from a mammalian host cell transformed with an expression vector containing a cDNA encoding the dual-function protein or its derivative, comprising culturing the mammalian host cell in a culture medium supplemented with dextran sulfate, wherein the dual-function protein comprises a fibroblast growth factor 21 (FGF21) mutant protein; a biologically active protein, or a mutant or fragment thereof; and an immunoglobulin Fc region, wherein the FGF21 mutant protein comprises substitutions of some amino acids in the amino acid sequence and amino acid mutations to reduce the immunogenicity of the FGF21 protein.

Description

[Technical Field]

[0001] Technical Field The present invention relates to methods for producing dual function proteins, including biologically active proteins and fibroblast growth factor 21 (FGF21) mutant proteins. [Background technology]

[0002] Background technology When animal cells are used to produce recombinant proteins, problems can occur in which certain regions of the target protein are cleaved by proteases secreted by the animal cells (host cells), causing heterogeneity, degradation, or inactivation of the recombinant protein. In addition, such cleavage of the expressed protein also leads to the problem of making it difficult to maintain "lot-to-lot" homogeneity during the production and purification process. For this reason, it is necessary to maintain proteases at low levels or suppress protease activity during the production of recombinant proteins.

[0003] As an alternative to this problem, production methods have been proposed in which inhibitors of serine, cysteine, aspartic acid, or aminopeptidases (e.g., aprotinin, bestatin, leupeptin, E-64, and pepstatin A) are added to the culture medium (see WO 1990-002175, EP 0,306,968, and US 5,851,800). However, the use of these inhibitors in commercial production is ineffective due to their cytotoxicity and the need for special efforts to prove that they are completely removed from the final product. In addition, among the conventional alternatives, a universal method applicable to all target proteins produced in host cells has not yet been found. Summary of the Invention

[0004] Description of the invention technical issues An object of the present invention is to provide a culture method for producing dual-function proteins, including biologically active proteins and FGF21 mutant proteins, with improved pharmacokinetic parameters, high stability, low potential for aggregation to form complexes, and reduced potential for immunogenicity.

[0005] solution According to one object of the present invention, there is provided a method for producing a recombinant dual function protein from a mammalian host cell transformed with an expression vector comprising a cDNA encoding the dual function protein or a derivative thereof, the method comprising culturing the mammalian host cell in a culture medium supplemented with dextran sulfate, wherein the dual function protein comprises a fibroblast growth factor 21 (FGF21) mutant protein; a biologically active protein, or a variant or fragment thereof; and an Fc region of an immunoglobulin, wherein the FGF21 mutant protein comprises at least one mutation selected from the group consisting of the following mutations (1)-(7): (1) substitution of amino acids with the amino acid sequence of EIRP (SEQ ID NO: 53) at positions 98-101 from the N-terminus of the wild-type FGF21 protein; (2) a substitution of amino acids at positions 170-174 from the N-terminus of the wild-type FGF21 protein with the amino acid sequence of TGLEAV (SEQ ID NO: 54); (3) a substitution of amino acids at positions 170-174 from the N-terminus of the wild-type FGF21 protein with the amino acid sequence of TGLEAN (SEQ ID NO: 55); (4) an amino acid substitution with amino acid N at position 170 from the N-terminus of the wild-type FGF21 protein; (5) an amino acid substitution with amino acid N at position 174 from the N-terminus of the wild-type FGF21 protein; (6) a substitution of an amino acid with amino acid E at position 180 from the N-terminus of the wild-type FGF21 protein, together with one or more of the above mutations (1) to (5); and (7) mutation of 1 to 10 amino acids to reduce the immunogenicity of wild-type FGF21 protein; The purpose is to provide a method.

[0006] Advantageous Effects of the Invention The production method of the present invention effectively prevents degradation of the target protein, thereby enabling stable production of the target protein. [Brief explanation of the drawings]

[0007] [Figure 1] Figure 1 shows the results of SDS-PAGE analysis of the culture supernatant of a cell line expressing a dual-function protein after suspension culture. We found that, over time, other proteins smaller than the uncleaved dual-function protein were also expressed.

[0008] [Figure 2] Figure 2 shows the results of SDS-PAGE analysis of culture supernatants after storage at various temperatures. Culture supernatants stored at 4°C or -20°C showed reduced cleavage of the dual-function protein compared with those stored at 37°C. This result indicated that cleavage of the dual-function protein was induced by proteases derived from host cells.

[0009] [Figure 3] Figure 3 shows the results of SDS-PAGE analysis of culture supernatants containing protease inhibitors after storage at 37°C. The addition of serine protease inhibitors significantly reduced the cleavage of the dual-function protein. This result suggests that the cleavage of the dual-function protein was induced by proteases derived from the host cells.

[0010] [Figure 4]Figure 4 shows the results of SDS-PAGE analysis of the culture supernatant after cell culture with dextran sulfate added to the culture medium. The effect of reducing the cleavage of the dual-function protein was not observed when dextran sulfate with a weight-average molecular weight of 1.6 kDa was added, but the cleavage of the dual-function protein was reduced when dextran sulfate with a weight-average molecular weight of 500 kDa was added.

[0011] [Figure 5] Figure 5 shows the results of SDS-PAGE analysis of culture supernatants after incubation with dextran sulfates of various weight-average molecular weights. When dextran sulfates with a weight-average molecular weight of 200 kDa or greater were added to the culture medium, the cleavage of the dual-function protein decreased.

[0012] [Figure 6] Figure 6 shows the results of SDS-PAGE of the culture supernatant after culturing in which various concentrations of dextran sulfate were added to the culture medium, and a graph of the results. When dextran sulfate was added at 200-1,000 mg / L, the cleavage of the dual-function protein was reduced. In addition, when the culture temperature was changed to 32°C during cultivation, the cleavage of the dual-function protein was more effectively prevented. DETAILED DESCRIPTION OF THE INVENTION

[0013] BEST MODE FOR CARRYING OUT THE INVENTION According to one object of the present invention, there is provided a method for producing a recombinant dual function protein from a mammalian host cell transformed with an expression vector comprising a cDNA encoding the dual function protein or a derivative thereof, the method comprising culturing the mammalian host cell in a culture medium supplemented with dextran sulfate, wherein the dual function protein comprises a fibroblast growth factor 21 (FGF21) mutant protein; a biologically active protein, or a variant or fragment thereof; and an Fc region of an immunoglobulin, wherein the FGF21 mutant protein comprises at least one mutation selected from the group consisting of the following mutations (1)-(7): (1) a substitution of amino acids at positions 98-101 from the N-terminus of the wild-type FGF21 protein with the amino acid sequence of EIRP (SEQ ID NO: 53) (hereinafter "EIRP"); (2) a substitution of amino acids at positions 170-174 from the N-terminus of the wild-type FGF21 protein with the amino acid sequence of TGLEAV (SEQ ID NO: 54) (hereinafter "TGLEAV"); (3) a substitution of amino acids at positions 170-174 from the N-terminus of the wild-type FGF21 protein with the amino acid sequence of TGLEAN (SEQ ID NO: 55) (hereinafter "TGLEAN"); (4) an amino acid substitution with amino acid N at position 170 from the N-terminus of the wild-type FGF21 protein (hereinafter, "G170N"); (5) an amino acid substitution with amino acid N at position 174 from the N-terminus of the wild-type FGF21 protein (hereinafter, "G174N"); (6) a substitution of an amino acid with amino acid E at position 180 from the N-terminus of the wild-type FGF21 protein (hereinafter "A180E"), together with one or more of the above mutations (1) to (5); and (7) mutation of 1 to 10 amino acids to reduce the immunogenicity of wild-type FGF21 protein; The purpose is to provide a method.

[0014] FGF21 mutant proteins The wild-type FGF21 protein, a hormone known to play an important role in glucose and lipid homeostasis, may be derived from a mammal, such as a human, mouse, pig, monkey, etc., preferably a human. More preferably, the wild-type FGF21 protein may be a wild-type human FGF21 protein having the amino acid sequence shown in SEQ ID NO: 1.

[0015] Preferably, the mutations contained in the FGF21 mutant protein may be any one of the mutations EIRP, TGLEAV, TGLEAN, G170N, and G174N; a combination of any one of the mutations TGLEAV, TGLEAN, G170N, and G174N and the mutation EIRP; a combination of any one of the mutations EIRP, TGLEAV, TGLEAN, G170N, and G174N and the mutation A180E; or a combination of any one of the mutations TGLEAV, TGLEAN, G170N, and G174N, an mutation EIRP, and the mutation A180E.

[0016] EIRP refers to a mutation in which LLLE, the amino acid at positions 98-101 from the N-terminus of the wild-type FGF21 protein, is replaced with EIRP. TGLEAV refers to a mutation in which GPSQG, the amino acid at positions 170-174 from the N-terminus of the wild-type FGF21 protein, is replaced with TGLEAV. TGLEAN refers to a mutation in which GPSQG, the amino acid at positions 170-174 from the N-terminus of the wild-type FGF21 protein, is replaced with TGLEAN. G170N refers to a mutation in which G, the amino acid at position 170 from the N-terminus of the wild-type FGF21 protein, is replaced with N. G174N refers to a mutation in which G, the amino acid at position 174 from the N-terminus of the wild-type FGF21 protein, is replaced with N.

[0017] Furthermore, the FGF21 mutant protein may have a structure in which 1 to 10 amino acids at the N-terminus or C-terminus are deleted compared to the wild-type FGF21 protein. More preferably, the FGF21 mutant protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 6 to 23. Even more preferably, the FGF21 mutant protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 6 to 23, and may further have a structure in which 1 to 10 amino acids at the N-terminus or C-terminus are deleted compared to the wild-type FGF21 protein.

[0018] In the dual-function protein, the amino acid residue N of the FGF21 mutant protein introduced by mutation can be glycosylated.

[0019] biologically active proteins The biologically active protein may be one selected from the group consisting of insulin, C-peptide, leptin, glucagon, gastrin, gastric inhibitory polypeptide (GIP), amylin, calcitonin, cholecystokinin, peptide YY, neuropeptide Y, bone morphogenetic protein-6 (BMP-6), bone morphogenetic protein-9 (BMP-9), oxyntomodulin, oxytocin, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), irisin, fibronectin type III domain-containing protein 5 (FNDC5), apelin, adiponectin, C1q and tumor necrosis factor-related protein (CTRP family), resistin, visfatin, omentin, retinol-binding protein-4 (RBP-4), glicentin, angiopoietin, interleukin-22 (IL-22), exendin-4, and growth hormone. Preferably, the biologically active protein may be one selected from GLP-1, a variant thereof, and exendin-4.

[0020] The GLP-1 protein is an incretin hormone consisting of 31 amino acids that is secreted by L cells in the intestinal tract, such as in response to stimulation by food. For example, the GLP-1 protein can be represented by the amino acid sequence of SEQ ID NO:29.

[0021] The GLP-1 variant may be represented, for example, by the amino acid sequence of any of SEQ ID NOs: 30 to 33.

[0022] Fc region of immunoglobulin As used herein, the terms "Fc region," "Fc fragment," or "Fc" refer to a protein that contains immunoglobulin heavy chain constant region 1 (CH1), heavy chain constant region 2 (CH2), and heavy chain constant region 3 (CH3), but does not contain immunoglobulin heavy and light chain variable regions and light chain constant region 1 (CL1). Furthermore, as used herein, the term "Fc region variant" refers to one prepared by substituting a portion of the amino acids in the Fc region or by combining various types of Fc regions.

[0023] The Fc region of an immunoglobulin may be the complete Fc region constituting an antibody, a fragment thereof, or an Fc region variant. Furthermore, the Fc region may include a molecule in the form of a monomer or a polymer, and may further include the hinge region of the heavy chain constant region. The Fc region variant may be modified to prevent cleavage at the hinge region. Furthermore, the hinge sequence of the Fc may have substitutions in several amino acids to reduce antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In addition, a portion of the amino acid sequence of the Fc hinge sequence may be substituted to inhibit rearrangement of the Fab region. The lysine residue at the C-terminus of the Fc may be removed.

[0024] Preferably, the immunoglobulin Fc region can be any one of IgG1, IgG2, IgG3, IgG4, and IgD Fc regions, or a hybrid Fc region that is a combination thereof. Furthermore, the hybrid Fc region can contain an IgG4 region and an IgD region. Furthermore, the hybrid Fc region can contain a portion of the hinge sequence and CH2 of an IgD Fc and the CH2 and CH3 sequences of an IgG4 Fc.

[0025] In addition, the Fc fragment of the present invention may be in the form of wild-type glycosylated chains, more glycosylated than wild-type, less glycosylated than wild-type, or deglycosylated chains. Increase, decrease, or elimination of glycosylated chains can be achieved by conventional methods known in the art, such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms.

[0026] Preferably, the immunoglobulin Fc region may be represented by an amino acid sequence selected from SEQ ID NOs: 24 to 28.

[0027] Dual-function proteins The dual function protein may comprise a biologically active protein, an immunoglobulin Fc region, and an FGF21 mutant protein, linked in this order from N-terminus to C-terminus. Furthermore, the dual function protein may comprise an FGF21 mutant protein, an immunoglobulin Fc region, and a biologically active protein, linked in this order from N-terminus to C-terminus. Preferably, the dual function protein may comprise a GLP-1 mutant protein, an immunoglobulin Fc region, and an FGF21 mutant protein, linked in this order from N-terminus to C-terminus. Furthermore, the dual function protein may comprise an FGF21 mutant protein, an immunoglobulin Fc region, and a GLP-1 mutant protein, linked in this order from N-terminus to C-terminus.

[0028] Linker Additionally, the dual function protein may further comprise a linker.

[0029] The dual-function protein may be in a form in which the FGF21 mutant protein is directly linked to the N-terminus or C-terminus of an immunoglobulin Fc region, or in which the FGF21 mutant protein is linked to an immunoglobulin Fc region via a linker.

[0030] In such cases, the linker can be linked to the N-terminus, C-terminus, or free base of the Fc fragment, or to the N-terminus, C-terminus, or free base of the FGF21 mutant protein. When the linker is a peptide linker, the linkage can occur in any region. For example, the linker can be linked to the C-terminus of an immunoglobulin Fc region and the N-terminus of an FGF21 mutant protein to form a fusion protein of an immunoglobulin Fc region and an FGF21 mutant protein. Furthermore, the dual-function protein of the present invention can be in a form in which a biologically active protein is linked to the N-terminus of the immunoglobulin Fc region of the fusion protein.

[0031] When the linker and Fc are expressed separately and then linked, the linker can be a cross-linking agent known in the art. Examples of cross-linking agents include, but are not limited to, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, such as 4-azidosalicylic acid and imidoesters including disuccinimidyl esters, such as 3,3'-dithiobis(succinimidyl propionate), and bifunctional maleimides, such as bis-N-maleimido-1,8-octane.

[0032] Furthermore, the linker may be a peptide, preferably a peptide consisting of 10 to 30 amino acid residues.

[0033] Furthermore, an alanine may be further attached to the end of the linker. Preferably, the linker may be a peptide having an amino acid sequence represented by any one of SEQ ID NOs: 2 to 5.

[0034] The dual function protein may be in the form of a dimer or multimer of FGF21 mutant proteins, in which one or more FGF21 mutant proteins are linked to each other, linked to an immunoglobulin Fc region.Furthermore, the dual function protein may be in the form of a dimer or multimer in which two or more immunoglobulin Fc regions are linked, and have FGF21 mutant proteins linked to the immunoglobulin Fc regions.

[0035] mammalian host cells The mammalian host cell can be any animal cell capable of expressing the recombinant dual-function protein, preferably an animal cell that facilitates the isolation of targeted transformed cells. Specifically, the mammalian host cell can be an immortalized hybridoma cell, an NS / 0 myeloma cell, a 293 cell, a Chinese hamster ovary cell (CHO cell), a HeLa cell, a CAP cell (human amniotic fluid-derived cell), or a COS cell.

[0036] Dextran sulfate As a result of applying a protease inhibitor to the cell culture of the dual-function protein of the present invention, the effect of preventing the cleavage of the dual-function protein by proteases derived from the host cells was insufficient.

[0037] The dextran sulfate may have a weight-average molecular weight of 20-5,000 kDa. Specifically, the dextran sulfate may have a weight-average molecular weight of 200-5,000 kDa.

[0038] In addition, the culture medium may contain dextran sulfate at a concentration of 0.01-10 g / L. Specifically, the culture medium may contain dextran sulfate at a concentration of 0.1-10 g / L, or 0.1-1 g / L.

[0039] culture The culturing may include the steps of primary culturing mammalian host cells in a culture medium supplemented with dextran sulfate at 34-37°C and subculturing the primary culture medium at 28-33°C. Specifically, the primary culture may be performed for 24-144 hours, and the subculturing may be performed at 31-33°C.

[0040] The dual function protein is a polypeptide in which GLP-1 and FGF21 variants, biologically active proteins, are fused to the Fc region of an immunoglobulin, and are expressed in intact form when produced by animal cell culture and exhibit activity as a composition for preventing or treating hepatitis, liver fibrosis, and cirrhosis.

[0041] Mode of the Invention Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to examples. However, the examples of the present invention can be modified in many different forms, and the scope of the present invention should not be limited to the examples described herein. [Example]

[0042] [Example] Preparation Example 1. Preparation of host cells for expression of dual-function proteins 1-1: Preparation of expression vectors for the expression of dual-function proteins The position, sequence information, target and expected effect of each mutation introduced into the FGF21 protein are listed in Table 1 below (in Table 1, N indicates glycosylated asparagine (N). Additionally, FGF21 mutant proteins containing the mutations described in Table 1 are listed in Table 2 below.

[0043] [Table 1]

[0044] [Table 2]

[0045] The GLP-1 variant protein sequences are shown in Table 3 below, and the Fc-fused GLP-1 variant protein sequences are shown in Table 4.

[0046] [Table 3]

[0047] [Table 4]

[0048] In Table 4, HyFc5 represents SEQ ID NO: 27, and HyFc40 represents SEQ ID NO: 28.

[0049] Furthermore, the sequences of dual function proteins comprising a GLP-1 mutant protein and an FGF21 mutant protein are listed below in Table 5. Each dual function protein comprises a GLP-1 mutant protein, an immunoglobulin Fc region, a linker, and an FGF21 mutant protein linked in this order from N-terminus to C-terminus.

[0050] [Table 5]

[0051] Specifically, the nucleotide sequences encoding each of the dual-function proteins were synthesized based on the amino acid sequences of each protein after consultation with Bioneer Corporation (Korea). NheI and NotI restriction enzyme sequences were added to the 5' and 3' ends of the nucleotide sequences encoding each of the dual-function proteins, and an initiation codon for protein translation and a leader sequence (SEQ ID NO: 56, MDAMLRGLCCVLLLCGAVFVSPSHA) enabling secretion of the expressed protein outside the cell were inserted next to the restriction enzyme sequence at the 5' end. A stop codon was inserted next to the nucleotide sequence encoding each of the FGF21 mutant proteins. The nucleotide sequences encoding each of the dual-function proteins were cloned into the pTrans-empty expression vector using the two restriction enzymes NheI and NotI. The pTrans-empty expression vector, which contains a CMV promoter, a pUC-derived replication origin, an SV40-derived replication origin, and an ampicillin-resistance gene, was purchased from CEVEC Pharmaceuticals (Germany).

[0052] 1-2: Construction of plasmid DNA for expression of dual-function proteins To obtain large amounts of plasmid DNA for expression, E. coli was transformed with each of the expression vectors constructed in Preparation Example 1-1. E. coli cells with weakened cell walls via heat shock were transformed with each expression vector, and the transformants were plated on LB plates to obtain colonies. The resulting colonies were inoculated into LB medium and cultured at 37°C for 16 hours to obtain 100 ml of E. coli cultures containing the respective expression vectors. The resulting E. coli was then centrifuged to remove the culture medium, and P1, P2, and P3 solutions (QIAGEN, Cat. No. 12963) were then added to disrupt the cell walls, thereby obtaining DNA suspensions from which proteins and DNA were separated. Plasmid DNA was purified from the resulting DNA suspension using a Qiagen DNA purification column. The eluted plasmid DNA was identified by agarose gel electrophoresis, and the concentration and purity were measured using a nanodrop device (Thermo Scientific, Nanodrop Lite). The DNA obtained in this manner was used for expression.

[0053] 1-3: Production of transformed host cells for expression of dual-function proteins CHO DG44 cells (Chinese hamster ovary cells) were transformed with each of the plasmid DNAs isolated in Preparation Example 1-2 using FreeStyleMAX (Invitrogen, Cat. No. 16447-100). The transformed Chinese hamster ovary cells were seeded in medium (CD OptiCHO, Gibco, Cat. No. 12681-011) and cultured in an incubator under conditions of 8% CO2 and 37°C. Surviving cells were selected and cultured by repeated passage. Finally, the selected cells were selected as single clones by limiting dilution in a 96-well plate.

[0054] Experimental Example 1: Suspension culture for expression of dual-function proteins and degradation phenomenon A CHO cell line transformed with substance code DFD112 (SEQ ID NO: 51) of Preparation Example 1-3 was cultured in suspension in CD OptiCHO medium supplemented with 8 mM GlutaMAX (working volume 30 ml / 125 ml flask, 37°C, 8% CO2, 120 rpm). The culture supernatant was then stored at three different temperatures (37°C, 4°C, or -20°C) for 3 days, and the extent of proteolysis was assessed by SDS-PAGE (4-12% Bis-Tris, non-reducing conditions) analysis of the culture supernatant. The results of the SDS-PAGE analysis are shown in Figures 1 and 2.

[0055] As shown in Figure 1 , we found that proteins smaller than the target protein (85 to 110 kDa) were expressed along with the uncleaved (intact) target protein during cell culture to produce the dual-function protein.

[0056] As shown in Figure 2, the culture supernatants stored at 4°C and -20°C contained fewer small proteins resulting from cleavage of the dual-function protein than the culture supernatants stored at 37°C. Therefore, we found that the degradation of the dual-function protein was caused by proteases secreted from host cells present in the culture supernatant.

[0057] Experimental Example 2. Detection of protease inhibitors involved in dual-function protein degradation To examine the category of proteases involved in the degradation of the dual-function protein identified in Experimental Example 1, various protease inhibitors were added to the culture supernatant of Experimental Example 1, incubated at 37°C for 3 days, and then subjected to SDS-PAGE analysis. The protease inhibitors used herein are listed in Table 6, and the results of SDS-PAGE analysis are shown in Figure 3.

[0058] [Table 6]

[0059] As shown in Figure 3, we found that the degradation of the dual-function protein was reduced in culture supernatants treated with serine protease inhibitors, such as AEBSF, antipain, leupeptin, benzamidine-HCl, and aprotinin, indicating that the degradation of the dual-function protein was caused by proteases derived from the host cells.

[0060] Example 1. Dextran sulfate treatment To prevent cleavage of the dual-function protein during cell culture, the transformed CHO cell lines of Preparations 1-3 were cultured in suspension in CD Dynamis medium (Gibco, cat. No. A2661501) supplemented with 6 mM glutamine for 7 days (working volume 30 mL / 125 mL flask, 37°C, 8% CO2, 120 rpm). For the suspension culture, dextran sulfate (weight-average molecular weight: 1.6 kDa or 500 kDa) was added to the culture medium at a concentration of 200 mg / L, and the culture was performed at 32°C using a low-temperature conversion and fed-batch culture method. The culture supernatant was then analyzed by SDS-PAGE (4-12% Bis-Tris, non-reducing conditions). The results of the SDS-PAGE analysis and a schematic diagram are shown in Figure 4. In Figure 4, w / o and Lane 1 are controls, 500 kDa and Lane 2 are culture supernatants treated with 500 kDa dextran sulfate, and 1.6 kDa and Lane 3 are culture supernatants treated with 1.6 kDa dextran sulfate.

[0061] As shown in Figure 4, we found that the cleavage phenomenon of the dual-function protein was effectively inhibited when dextran sulfate with a weight-average molecular weight of 500 kDa was added to the culture medium.

[0062] Example 2. Effect of molecular weight on dextran sulfate The effective concentration range of added dextran sulfate, identified in Example 1 as having protective effect against cleavage of dual-function proteins during cell culture, was examined.

[0063] Specifically, except for adjusting the weight-average molecular weight (1.6 kDa, 8 kDa, or 200 kDa) and concentration (100 mg / L, 200 mg / L, or 500 mg / L) of the added dextran sulfate, the culture was carried out under the same conditions as in Example 1. The culture supernatant was then analyzed by SDS-PAGE (4-12% Bis-Tris, non-reducing conditions). The results of the SDS-PAGE analysis and a graph thereof are shown in FIG. 5.

[0064] As shown in Figure 5, the cleavage phenomenon of the dual-function protein was significantly reduced when dextran sulfate with a molecular weight of 200 kDa or more was added to the culture medium at concentrations of 100 to 500 mg / L.

[0065] Example 3. Evaluation of culture conditions for prevention of dual-function protein degradation Culture conditions were tested to maximize the effectiveness of preventing cleavage of dual function proteins by dextran sulfate as identified in Examples 1 and 2.

[0066] Specifically, the culture was carried out under the same conditions as in Example 1, except that 500 kDa dextran sulfate was added at concentrations ranging from 0 mg / L to 1,000 mg / L. An experimental group was included in which the culture temperature was changed to 32°C on day 4 of culture (see Table 7 below). The culture supernatant was then analyzed by SDS-PAGE (4-12% Bis-Tris, non-reducing conditions), and the results of the SDS-PAGE analysis and a graph thereof are shown in Figure 6.

[0067] [Table 7]

[0068] As shown in Figure 6, the culture supernatant from the culture treated with dextran sulfate having a weight-average molecular weight of 500 kDa showed significantly reduced cleavage of the dual-function protein compared to the culture supernatant from the culture without dextran sulfate treatment. Furthermore, when the temperature was changed during the culture, the cleavage of the dual-function protein was more effectively inhibited.

Claims

1. 1. A method for producing a recombinant dual function protein from a mammalian host cell transformed with an expression vector comprising a cDNA encoding the dual function protein or a derivative thereof, the method comprising culturing the mammalian host cell in a culture medium supplemented with dextran sulfate, wherein the dual function protein comprises a fibroblast growth factor 21 (FGF21) mutant protein; a biologically active protein; and an Fc region of an immunoglobulin, wherein the FGF21 mutant protein (1) substitution of amino acids at positions 98-101 from the N-terminus of the wild-type FGF21 protein with the amino acid sequence of EIRP (SEQ ID NO: 53); and (6) an amino acid substitution with amino acid E at position 180 from the N-terminus of the wild-type FGF21 protein; and (2) a substitution of an amino acid with the amino acid sequence of TGLEAV (SEQ ID NO: 54) at positions 170-174 from the N-terminus of the wild-type FGF21 protein; (3) a substitution of an amino acid with the amino acid sequence of TGLEAN (SEQ ID NO: 55) at positions 170-174 from the N-terminus of the wild-type FGF21 protein; (4) a substitution of an amino acid with amino acid N at position 170 from the N-terminus of the wild-type FGF21 protein; and (5) a substitution of an amino acid with amino acid N at position 174 from the N-terminus of the wild-type FGF21 protein. Including, Biologically active proteins include insulin, C-peptide, leptin, glucagon, gastrin, gastric inhibitory polypeptide (GIP), amylin, calcitonin, cholecystokinin, peptide YY, neuropeptide Y, bone morphogenetic protein-6 (BMP-6), bone morphogenetic protein-9 (BMP-9), oxyntomodulin, oxytocin, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), irisin, and fibronectin type III domain-containing protein. 5 (FNDC5), apelin, adiponectin, C1q and tumor necrosis factor-related protein (CTRP family), resistin, visfatin, omentin, retinol-binding protein-4 (RBP-4), glicentin, angiopoietin, interleukin-22 (IL-22), exendin-4 and growth hormone, and a variant of GLP-1 having an amino acid sequence represented by any one of SEQ ID NOs: 30 to 33; and The wild-type FGF21 protein has the amino acid sequence set forth in SEQ ID NO:1, The dual function protein comprises the biologically active protein, the Fc region of the immunoglobulin, and the FGF21 mutant protein, linked in this order from N-terminus to C-terminus. method.

2. 2. The method of claim 1, wherein the dextran sulfate has an average molecular weight of 20-5,000 kDa.

3. 10. The method of claim 1, wherein the culture medium has dextran sulfate at a concentration of 0.01-10 g / L.

4. To cultivate Primary culturing mammalian host cells at 34-37°C in a culture medium supplemented with dextran sulfate; and Subculture of the primary culture medium at 28-33°C The method of claim 1 , comprising:

5. The method of claim 4, wherein the primary culture is carried out for 24 to 144 hours.

6. The method according to claim 4, wherein the subculture is carried out at 31-33°C.

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