Process for producing polypeptide
View Patent ↗The present invention relates to a process for producing a desired polypeptide using rat cells. Specifically, the present invention relates to a process for producing the polypeptide which comprises culturing rat cells such as YB2/3HL.P2.G11.16Ag.20 (hereinafter referred to as YB2/0), preferably rat cells to which a recombinant DNA comprising DNA encoding a desired polypeptide such as an immunologically functional molecule is introduced, in a medium which does not contain serum (hereinafter referred to as a serum-free medium). Among the desired polypeptides obtained by the process of the present invention, an antibody obtained by using a transformant of YB2/0 has a high antibody-dependent cell-mediated cytotoxic activity (hereinafter sometimes referred to as ADCC activity) and is useful as a pharmaceutical agent.
1. A process for producing a polypeptide which comprises:
(a) selecting a rat cell line capable of adapting in a serum-free medium
(b) culturing the rat cell line selected in step (a) in a serum-free medium while an insulin concentration in the culture is maintained at 10 mg/l or above, followed by culturing while an insulin concentration in the culture is maintained at 20 mg/l or below, and
(c) recovering the desired polypeptide from the culture,
wherein the step (a) is carried out by inoculating a rat cell line into a serum-free medium at the cell density of 1×10 5 to 10×10 5 cells/ml of serum free medium and selecting a rat cell line that reaches a cell density of 10×10 5 to 40×10 5 cells/ml of serum-free medium.
2. The process according to claim 1 , wherein the rat cell line adapted to a serum-free medium is a rat cell line which can be subcultured in a serum-free medium for two months or more.
3. The process according to claim 1 , wherein the rat cell is a myeloma cell or a hybrid cell derived from a myeloma cell.
4. The process according to claim 1 , wherein the rat cell is YB2/3HL.P2.G11.16Ag.20 (ATCC CRL 1662).
5. The process according to claim 1 , wherein the cell is a cell to which a recombinant DNA comprising DNA encoding the desired polypeptide is introduced.
6. The process according to claim 1 , wherein the culturing is carried out by batch culture, fed-batch culture or perfusion culture.
7. The process according to claim 1 , comprising adding at least one member selected from the group consisting of a nutrient factor and a physiologically active substance to the medium during the culturing.
8. The process according to claim 7 , wherein the nutrient factor is at least one member selected from the group consisting of glucose, an amino acid and a vitamin.
9. The process according to claim 1 , wherein the desired polypeptide is an immunologically functional molecule.
10. The process according to claim 9 , wherein the immunologically functional molecule is a protein or a peptide.
11. The process according to claim 10 , wherein the protein or peptide is an antibody, an antibody fragment or a fusion protein comprising an antibody Fc region.
12. The process according to claim 11 , wherein the antibody is an antibody recognizing a tumor-related antigen, an antibody recognizing an allergy- or inflammation-related antigen, an antibody recognizing a circulatory disease-related antigen, an antibody recognizing an autoimmune disease-related antigen, or an antibody recognizing a viral or bacterial infection-related antigen.
13. The process according to claim 12 , wherein the antibody recognizing a tumor-related antigen is an anti-GD2 antibody, an anti-GD3 antibody, an anti-GM2 antibody, an anti-HER2 antibody, an anti-CD52 antibody, an anti-MAGE antibody, an anti-basic fibroblast growth factor antibody, an anti-basic fibroblast growth factor receptor antibody, an anti-FGF8 antibody, an anti-FGF8 receptor antibody, an anti-insulin-like growth factor antibody, an anti-PMSA antibody, an anti-vascular endothelial cell growth factor antibody, or an anti-vascular endothelial cell growth factor receptor antibody; the antibody recognizing an allergy- or inflammation-related antigen is an anti-interleukin 6 antibody, an anti-interleukin 6 receptor antibody, an anti-interleukin 5 antibody, an anti-interleukin 5 receptor antibody, an anti-interleukin 4 antibody, an anti-interleukin 4 receptor antibody, an anti-tumor necrosis factor antibody, an anti-tumor necrosis factor receptor antibody, an anti-CCR4 antibody, an anti-chemokine antibody, or an anti-chemokine receptor antibody; the antibody recognizing a circulatory disease-related antigen is an anti-GpIIb/IIIa antibody, an anti-platelet-derived growth factor antibody, an anti-platelet-derived growth factor receptor antibody, or an anti-blood coagulation factor antibody; the antibody recognizing an autoimmune disease-related antigen is an anti-auto-DNA antibody; and the antibody recognizing a viral or bacterial infection-related antigen is an anti-gp120 antibody, an anti-CD4 antibody, an anti-CCR4 antibody, or an anti-verotoxin antibody.
14. The process according to claim 12 , wherein the antibody is an anti-GD3 human chimeric antibody, a humanized anti-interleukin 5 receptor a chain antibody, or an anti-GM2 human CDR-grafted antibody.
15. The process according to claim 1 , wherein the rat cell line can be cultured in a serum-free medium such that 90% or more of the cells of the original culture remain viable throughout said culturing.
16. The process of claim 1 wherein said culturing of step (b) comprises
culturing the rat cell line in a serum-free medium while an insulin concentration in the culture is maintained at 20 mg/l or above, followed by culturing while an insulin concentration in the culture is maintained at 10 mg/l or below.
17. The process of claim 1 wherein said culturing of step (b) comprises
culturing the rat cell line in a serum-free medium while an insulin concentration in the culture is maintained at 10 mg/l or above, followed by culturing while an insulin concentration in the culture is maintained at 10 mg/l or below.
18. The process of claim 1 wherein said culturing of step (b) comprises
culturing the rat cell line in a serum-free medium while an insulin concentration in the culture is maintained at 10 mg/l or above, followed by culturing while an insulin concentration in the culture is maintained at 5 mg/l or below.