Method for increasing expression of active tumor necrosis factor receptor family member-Ig fusion proteins
View Patent ↗Methods for high level expression of active lymphotoxin-β receptor immunoglobulin chimeric proteins and their purification.
1. A method for increasing expression of active tumor necrosis factor (TNF) receptor family member-Ig Fc fusion proteins and minimizing expression of inactive TNF receptor family member-Ig Fc fusion proteins comprising culturing a mammalian host cell transformed with DNA molecule encoding a desired TNF receptor-Ig fusion protein in a mammalian cell culture having a minimum temperature of about 27° C. to a maximum temperature of less than or equal to 30° C., wherein the transformed host cell is first cultured at a temperature of about 33° C. to about 37° C. for a period of time sufficient to allow growth of said host cell, and wherein the active TNF receptor family member is involved in immune regulation and comprises 2-4 copies of canonical TNF family cysteine-rich domains.
2. The method of claim 1 wherein the TNF receptor family member is a lymphotoxin-β receptor, TNFR-55, TNFR-75, herpes virus entry mediator (HVEM) or a ligand-binding portion thereof.
3. The method of claim 2 , wherein the TNF receptor family member is lymphotoxin-β receptor or a ligand-binding portion thereof.
4. The method of any one of claims 1 - 3 , further comprising the step of recovering the active TNF receptor family member-Ig Fc fusion proteins from the culture.
5. The method of claim 4 , wherein the active TNF receptor family members are recovered by hydrophobic interaction chromatography.
6. The method of claim 4 , wherein the active TNF receptor family member-Ig Fc fusion proteins are recovered based on their ability to bind an antibody that binds directly to the ligand binding region of the TNF receptor family member and exclusively recognizes the active form of the TNF receptor family member-Ig Fc fusion protein.
7. The method of claim 1 , wherein the mammalian host cell is a CHO cell.
8. The method of claim 1 , wherein the Ig Fc region is human.
9. The method of claim 1 , wherein the Ig Fc region is of an IgG1 isotype.