Methods for expressing ADAMTS proteins in cell culture medium supplemented with zinc
AssigneesBaxter International Inc., Baxter Healthcare S.A.
InventorsLeopold Grillberger, Alexandra Spenger, Meinhard Hasslacher, Rana Grillberger, Manfred Reiter
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Abstract
The present invention provides culture mediums that are useful for the expression of ADAMTS proteins, such as ADAMTS13. Methods for the expression and purification of ADAMTS proteins are also provided. In some embodiments, the mediums and methods of the invention are useful for the expression of ADAMTS proteins having high specific activities. Also provided are ADAMTS, e.g., ADAMTS13, protein compositions with high specific activities, which are expressed and purified according to the methods provided herein.
Claims30
A method for expressing a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) protein, the method comprising culturing a cell harboring a nucleic acid encoding an ADAMTS protein in a culture medium comprising at least 3 μM zinc,
- wherein the ADAMTS protein expressed according to the method has a greater specific activity as compared to an ADAMTS protein expressed in culture medium comprising 1.5 μM zinc.
The method of 15, wherein the specific activity of the ADAMTS13 protein in the culture medium is at least 600 U per mg ADAMTS13 protein.
A method for producing an ADAMTS composition, the method comprising the steps of:
- (a) culturing a cell harboring a nucleic acid encoding an ADAMTS protein in an animal protein-free culture medium comprising at least 3 μM zinc;
- (b) removing a fraction of the supernatant from the culture;
- (c) performing a filtration or centrifugation step to remove any residual cells;
- (d) performing an ultrafiltration step to concentrate the ADAMTS protein;
- and
- (e) performing a diafiltration step with a buffer comprising at least 0.5 μM zinc and at least 0.1 mM calcium;
- thereby preparing an ADAMTS composition.
Dependencies are inferred from the claim text.