Method of producing recombinant ADAMTS13 in cell culture

摘要

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 (NH4+) 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.

主权项

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 NH4+ 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 4.0×106 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.