Method of producing recombinant ADAMTS13 in cell culture
View Patent ↗Among other aspects, the present invention relates to cell culture conditions for producing high molecular weight vWF, in particular, highly multimericWF with a high specific activity and ADAMTS13 with a high specific activity. The cell culture conditions of the present invention can include, for example, a cell culture medium with an increased copper concentration and/or cell culture supernatant with a low ammonium (NH 4 + ) concentration. The present invention also provides methods for cultivating cells in the cell culture conditions to express high molecular weight vWF and rA13 having high specific activities.
1. A method for producing a recombinant ADAMTS 13 (rA13) composition, the method comprising the steps of:
(a) providing a protein free, chemically-defined, basal cell culture media;
(b) supplementing the basal cell culture media with copper to provide a final copper concentration of from 2 μg/L to 4 μg/L;
(c) providing one or more mammalian cells comprising a nucleic acid encoding a rA13 protein;
(d) culturing the one or more mammalian cells in the copper supplemented cell culture media such that rA13 is expressed and excreted from the cells into a culture supernatant, wherein the NH 4 + content of the cell culture supernatant is maintained at a concentration below 10 mM; and
(e) recovering at least a portion of the culture supernatant,
wherein the mammalian cell density is maintained at less than 4.0×10 6 cells per mL during the step of culturing the one or more cells; and
wherein at least 1500 Units FRETS-VWF73 activity per liter supplemented basal cell culture media per day is present in the recovered culture supernatant.
2. The method of claim 1 , wherein the mammalian cells are CHO cells.
3. The method of claim 1 , wherein culturing the one or more mammalian cells comprises batch cultivation of the cells.
4. The method of claim 1 , wherein culturing the one or more mammalian cells comprises continuous cultivation of the cells.
5. The method of claim 4 , wherein the continuous cultivation of mammalian cells is performed in chemostatic mode.
6. The method of claim 4 , wherein the continuous cultivation of mammalian cells is performed in perfusion mode.
7. The method of claim 1 , wherein the one or more mammalian cells is cultured in at least 100 L of the supplemented basal cell culture media.
8. The method of claim 1 , wherein the mammalian cell density is maintained at between 3.0×10 6 cells per mL and 4.0×10 6 cells per mL during the step of culturing the one or more cells.
9. The method of claim 1 , wherein at least 2000 Units FRETS-VWF73 activity per liter supplemented basal cell culture media per day is present in the recovered culture supernatant.
10. The method of claim 1 , wherein the NH 4 + content of the mammalian cell culture supernatant is maintained at a concentration below 5 mM.