METHOD FOR PRODUCING BIFUNCTIONAL PROTEINS AND THEIR DERIVATIVES

Claims

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

2. The method according to claim 1, wherein the biologically active protein is 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), glycentin, angiopoietin, interleukin-22 (IL-22), exendin-4 and growth hormone.

3. The method according to claim 1, wherein the bifunctional protein may comprise a biologically active protein, an Fc region of an immunoglobulin and a mutant FGF21 protein linked in such order from the N-terminus to the C-terminus.

4. The method according to claim 1, wherein dextran sulfate has an average molecular weight of 20 to 5000 kDa.

5. The method according to claim 1, wherein the culture medium contains dextran sulfate in a concentration of 0.01 to 10 g / l.

6. The method according to claim 1, wherein the culturing comprises: a primary cultivation stage of the mammalian host cell at 34-37°C in a culture medium supplemented with dextran sulfate; and the stage of secondary cultivation of the primary culture medium at 28-33°C.

7. The method according to claim 6, wherein the primary cultivation is carried out for 24 to 144 hours.

8. The method according to claim 1, wherein the secondary cultivation is carried out at a temperature of 31°C to 33°C.