Method of producing recombinant high molecular weight vWF 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 multimeric WF 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 cell culture supernatant comprising recombinant Von Willebrand Factor (rVWF), wherein at least 20% of the rVWF is present in a high molecular weight VWF multimer of more than 10 dimers, wherein the cell culture supernatant has been supplemented to provide a final copper concentration of at least 2.4 μg/L, and wherein the NH 4 + content of the cell culture supernatant is maintained at a concentration below 4 mM or below 10 mM.
2. The cell culture supernatant of claim 1 , wherein the supernatant contains at least 0.5 IU ristocetin cofactor activity per mL.
3. The cell culture supernatant of claim 1 , wherein the supernatant has a rVWF specific ristocetin cofactor activity of at least 30 mU/μg rVWF.
4. The cell culture supernatant of claim 1 , wherein the supernatant has a rVWF specific ristocetin cofactor activity of at least 40 mU/μg rVWF.
5. The cell culture supernatant of claim 1 , wherein the supernatant has a rVWF specific ristocetin cofactor activity of at least 50 mU/μg rVWF.
6. The cell culture supernatant of claim 1 , wherein the supernatant has a rVWF specific ristocetin cofactor activity of at least 60 mU/μg rVWF.
7. The cell culture supernatant of claim 1 , wherein the supernatant has a rVWF specific ristocetin cofactor activity of at least 70 mU/μg rVWF.
8. The cell culture supernatant of claim 1 , wherein the supernatant has a rVWF specific ristocetin cofactor activity of at least 80 mU/μg rVWF.
9. The cell culture supernatant of claim 1 , wherein at least 30% of the rVWF in the supernatant is present in a high molecular weight VWF multimer of more than 10 dimers.
10. The cell culture supernatant of claim 1 , wherein the rVWF is present in a high molecular weight VWF multimer of 14 to 22 dimers.
11. The cell culture supernatant of claim 1 , wherein the supernatant further comprises recombinant Factor VIII (rFVIII).
12. The cell culture supernatant of claim 11 , wherein the ratio of rVWF to rFVIII is at least 10:1.