Purification of cell culture derived alpha1 protease inhibitor
AssigneeGrifols, S.A.
InventorsDavid Ownby, Thomas P. Zimmerman, Jennifer A. Hunt, Charles Miller, Senthil Ranganathan, Tonny Dessources
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Abstract
Described herein are methods for purifying recombinant, cell culture derived alpha1-protease inhibitor and removing a colored species that co-purifies with the recA1PI protein. Also described are methods for reducing the iron in cell culture derived alpha1-protease inhibitor.
Claims26
A method of purifying cell culture derived human recombinant alpha1-protease inhibitor (recA1PI) from an aqueous solution comprising recA1PI, comprising:
- (a) performing a viral inactivation step on a solution containing recA1PI;
- (b) passing the virally inactivated solution through an anion exchanger so that recA1PI binds to the anion exchanger;
- (c) eluting recA1PI from the anion exchanger to obtain an anion exchange eluate containing recA1PI;
- (d) adding a reducing agent to the anion exchange eluate containing recA1PI to obtain a reducing solution, wherein the reducing agent is 10 mM cysteine;
- (e) incubating the reducing solution overnight at about room temperature;
- (f) passing the reducing solution through a hydrophobic interaction chromatography (HIC) resin so that recA1PI binds to the HIC resin;
- and
- (g) eluting recA1PI from the HIC resin to obtain an HIC eluate that contains recA1PI.
A method of purifying cell culture derived recombinant alpha1-protease inhibitor (recA1PI) from an aqueous solution comprising recA1PI, comprising:
- (a) performing a viral inactivation step on a solution containing recA1PI;
- (b) passing the virally inactivated solution through an anion exchanger so that recA1PI binds to the anion exchanger;
- (c) eluting recA1PI from the anion exchanger to obtain an anion exchange eluate containing recA1PI;
- (d) adding a reducing agent to the anion exchange eluate containing recA1PI to obtain a reducing solution;
- (e) incubating the reducing solution;
- (f) passing the reducing solution through a hydrophobic interaction chromatography (HIC) resin so that recA1PI binds to the HIC resin;
- and
- (g) eluting recA1PI from the HIC resin to obtain an HIC eluate that contains recA1PI,
- wherein the reducing agent is 2 mercaptoethanol (2-ME).
A method of purifying cell culture derived recombinant alpha′ proteinase inhibitor (recA1PI) comprising incubating a solution comprising recA1PI with 10 mM cysteine overnight at about room temperature and then separating the recA1PI from the reducing agent.
Dependencies are inferred from the claim text.