Substantially animal protein-free recombinant furin and methods for producing the same
View Patent ↗The present invention relates to recombinant furin (rFurin) and methods for producing rFurin. More specifically, the invention relates to substantially animal protein-free rFurin and methods for producing substantially animal protein-free rFurin.
1. A composition comprising substantially animal protein-free recombinant furin lacking transmembrane and cytoplasmic domains comprising a specific activity of at least or about 400 U/μg protein and host cell protein at a concentration less than about 1.0 ng protein/U furin activity, or host cell DNA at a concentration less than about 0.5 pg DNA/U furin activity, and essentially lacking contaminating proteins from serum.
2. The composition of claim 1 comprising recombinant furin at an activity of at least or about 10000 U furin/ml.
3. The composition of claim 1 comprising recombinant furin at an activity of at least or about 450 U/μg.
4. A method of making the composition of claim 1 comprising growing a host cell transformed or transfected with a polynucleotide encoding secreted recombinant furin lacking transmembrane and cytoplasmic domains in serum-free medium in a repeated fed batch process under conditions that permit secretion of the furin into the medium, binding the secreted furin on a multimodal cation exchange resin to remove host cell protein, host cell DNA, or a combination thereof; and recovering the secreted recombinant furin from the multimodal cation exchange resin.
5. The method of claim 4 comprising the step of adapting the host cell to grow in medium with increasingly lower concentrations of serum until all serum is removed from the medium.
6. The method of claim 4 comprising transferring the host cell from growth in medium comprising serum to growth in serum-free medium.
7. The method of claim 5 or 6 , wherein the host cell is a Chinese Hamster Ovary (CHO) cell.
8. A method of processing a pro-protein comprising a furin cleavage site comprising the step of contacting the pro-protein comprising the furin cleavage site with the composition of claim 1 under conditions to cleave a pro-peptide from the pro-protein to form a mature protein.
9. The method of claim 8 , wherein the mature protein is von Willebrand Factor.
10. The method of claim 8 , wherein the mature protein is Factor VIII.
11. The composition of claim 1 , wherein the host cell is a Chinese Hamster Ovary (CHO) cell.
12. The composition of claim 1 comprising recombinant furin at an activity of at least or about 500 U/μg.