Patent EA-001215-B1 (EA)
Title: Process for controlling the amount of sialic acid presenton an oligosaccharide side chaun of a glycoprotein, process for producing chimeric glycoprotein, preparation, comprising chimeric glycoprotein, and therapeutic composition
Inventor: ETCHEVERRY TINA
IPC: C12N15/09
Filed: 1996-06-06
Abstract: 1. A process for controlling the amount of sialic acid present on an oligosaccharide side chain of a glycoprotein produced by mammalian cell culture, by changing specific productivity of the culture, which comprises culturing the mammalian host cell in a production phase of the culture which is characterized by: i) adding in an intermediate phase an alkanoic acid having 3-6 carbon atoms or a salt thereof to the cell culture at a concentration of about 0.1 mM to about 20 mM: and ii) maintaining the osmolality of the cell culture at about 250 to about 600 mOsm, setting the culture temperature of about 30 to 35 degree C, and maintaining said parameters in a step of glycoprotein production. 2. The process according to claim 1 wherein for increasing the amount of sialic acid present on the oligosaccharide side chain of the glycoprotein the specific productivity of the cell culture is decreased by the following way: i) adding in an intermediate phase an alkanoic acid having 3-6 carbon atoms or a salt thereof to the cell culture at a concentration of about 0.1 mM to about 6 mM: and ii) maintaining the osmolality of the cell culture at about 300 to about 450 mOsm, setting the culture temperature of about 30 degree C to 35 degree C, and maintaining said parameters in a step of glycoprotein production. 3. The process of claim 2 in which the host cell is a CHO cell. 4. The process according to claim 3 wherein the alkanoic acid or salt thereof is sodium butyrate. 5. The process according to claim 4 wherein the glycoprotein produced by the cell culture is a mammalian glycoprotein. 6. The process according to claim 5 wherein the glycoprotein is a chimerical glycoprotein containing tumor necrosis factor receptor of a human and immunoglobulin G1. 7. The process of claim 6 in which the host cell is a dp12.CHO cell line transfected with the vector carrying the cDNA encoding a soluble type 1 tumor necrosis factor immunoglobulin G1 chimera (TNFR1-IgG1). 8. The process according to claim 1 wherein for decreasing the amount of sialic acid present on the oligosaccharide side chain of the glycoprotein the specific productivity of the cell culture is decreased by the following way: i) adding in an intermediate phase an alkanoic acid having 3-6 carbon atoms or a salt thereof to the cell culture at a concentration of about 6 mM to about 12 mM: and ii) maintaining the osmolality of the cell culture at about 450 to about 600 mOsm, setting the culture temperature of about 30 degree C to 35 degree C, and maintaining said parameters in a step of glycoprotein production. 9. The process of claim 8 in which the host cell is a CHO cell. 10. The process according to claim 9 wherein the alkanoic acid or salt thereof is sodium butyrate. 11. The process according to claim 10 wherein the glycoprotein produced the cell culture is a mammalian glycoprotein. 12. The process according to claim 11 wherein the glycoprotein is a tumor necrosis factor receptor-immunoglobulin G1. 13. The process of claim 12 in which the host cell is a dp12.CHO cell line cell transfected with the vector carrying the cDNA for a soluble type 1 tumor necrosis factor immunoglobulin G1 (TNFR1-IgG1). 14. A process for producing a chimeric immunoglobulin comprising a soluble type 1 tumor necrosis factor immunoglobulin G1 chimera (TNFR1-IgG1) and having a decreased amount of of sialic acid present on the oligosaccharide side chain, comprising the following steps: (a) culturing CHO cells expressing a TNFR-Ig chimera at a temperature of about 37 degree C under such conditions that maximum cell growth is achieved; (b) culturing the host cell in a production phase in the presence of sodium butyrate at a  concentration of about 6mM to about 12 mM; (c) maintaining the osmolality of the production phase at about 450-600 mOsm, setting the culture temperature of about 30 degree C to 35 degree C, and maintaining said parameters in a step of glycoprotein production, separate glycoprotein from the culture medium and purify. 15. A process for producing a chimeric immunoglobulin comprising a soluble type 1 tumor necrosis factor immunoglobulin G1 chimera (TNFR1-IgG1) and having an increase amount of of sialic acid present on the oligosaccharide side chain, comprising the following steps: (a) culturing mammalian host cells expressing a TNFR-Ig chimera at a temperature of about 37 degree C under such conditions that maximum cell growth is achieved; (b) culturing the host cell in a production phase in the presence of sodium butyrate at a  concentration of about 1mM to about 6 mM; (c) maintaining the osmolality of the production phase at about 300-450 mOsm, setting the culture temperature of about 30 degree C to 35 degree C, and maintaining said parameters in a step of glycoprotein production, separate glycoprotein from the culture medium and purify. 16. The process of claim 15 in which the host cell is a CHO cell. 17. The process of claim 16 in which the host cell is a dp 12.CHO line. 18. A preparation comprising the TNFR1-IgGl produced by the process of claim 17. 19. A therapeutic composition comprising the TNFR1-IgG1 produced by the process of claim 15 and a pharmaceutically acceptable excipient. 20. A TNFR1-IgG1 preparation comprising TNFR1-IgG1 molecules produced by the process ofclaim 14 or 15 wherein the TNFR1-IgG1 molecules have a molar ratio of sialic acid to protein of about 4-7. 21. A TNFR1-IgG1 preparation comprising TNFR1-IgG1 molecules produced by the process ofclaim 15 wherein the TNFR1-IgG1 molecules have about 1-2 moles of exposed N-acetylglucosamine residues per mole of TNFR1-IgG1 protein. 22. A TNFR1-IgG1 preparation comprising TNFR1-IgG1 molecules produced by the process of claim 15 wherein the TNFR1-IgG1 molecules have a molar ratio of sialic acid to N-acetylglucosamine of about 0.35 to about 0.5. 23. The TNFR1-IgGl preparation of claim 22 wherein the molar ration of sialic acid to N. acetylglucosamine is about 0.39 to about 0.45.