Purification of cell culture derived alpha1 protease inhibitor
Inventors
OWNBY, DAVID • Zimmerman, Thomas P. • Hunt, Jennifer A. • Miller, Charles • RANGANATHAN, SENTHIL • DESSOURCES, TONNY
Assignees
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Publication Number
US-9957315-B2
Publication Date
2018-05-01
Expiration Date
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.
Core Innovation
The invention is directed to purification of cell culture derived human recombinant alpha1-protease inhibitor (recA1PI) from an aqueous solution containing recA1PI. The disclosed workflow includes performing a viral inactivation step on a solution containing recA1PI, followed by passing the virally inactivated solution through an anion exchanger so that recA1PI binds, and eluting recA1PI to obtain an anion exchange eluate.
The invention further provides a reduction and polishing sequence in which a reducing agent is added to the anion exchange eluate containing recA1PI to obtain a reducing solution. The reducing solution is incubated, and recA1PI is then captured by passing the reducing solution through a hydrophobic interaction chromatography (HIC) resin so that recA1PI binds to the HIC resin, followed by eluting recA1PI from the HIC resin to obtain an HIC eluate.
The document describes a specific approach for reducing and/or removing a co-purifying yellow colored species/colorant associated with recA1PI. It also describes reducing iron content associated with recA1PI, and reports that the integrated sequence of reduction with chromatography diminishes the yellow color while improving activity recovery and reducing iron concentration in the product fraction.
Claims Coverage
The partial content includes three independent claims, each centered on purifying cell culture derived recombinant alpha1-protease inhibitor (recA1PI) using viral inactivation, chromatographic capture/elution steps, and incubation with a reducing agent, with claim-specific constraints related to the reducing agent and iron reduction.
Purifying recA1PI with viral inactivation, anion exchange capture, cysteine reduction, and HIC polishing
Performing a viral inactivation step on a solution containing recA1PI; passing the virally inactivated solution through an anion exchanger so that recA1PI binds to the anion exchanger; eluting recA1PI from the anion exchanger to obtain an anion exchange eluate; adding a reducing agent to the anion exchange eluate to obtain a reducing solution wherein the reducing agent is 10 mM cysteine; incubating the reducing solution; passing the reducing solution through a hydrophobic interaction chromatography (HIC) resin so that recA1PI binds to the HIC resin; and eluting recA1PI from the HIC resin to obtain an HIC eluate.
Purifying recA1PI with viral inactivation, anion exchange capture, 2-ME reduction, and HIC polishing
Performing a viral inactivation step on a solution containing recA1PI; passing the virally inactivated solution through an anion exchanger so that recA1PI binds to the anion exchanger; eluting recA1PI from the anion exchanger to obtain an anion exchange eluate; adding a reducing agent to the anion exchange eluate to obtain a reducing solution; incubating the reducing solution; passing the reducing solution through a hydrophobic interaction chromatography (HIC) resin so that recA1PI binds to the HIC resin; and eluting recA1PI from the HIC resin to obtain an HIC eluate, wherein the reducing agent is 2 mercaptoethanol (2-ME).
Incubating recA1PI with 10 mM cysteine and separating from the reducing agent
Purifying cell culture derived recombinant alpha1 proteinase inhibitor (recA1PI) comprising incubating a solution comprising recA1PI with 10 mM cysteine and then separating the recA1PI from the reducing agent.
Across the independent claims, the core inventive coverage is the combination of viral inactivation with chromatographic capture/elution on an anion exchanger and polishing by hydrophobic interaction chromatography, together with incubation of recA1PI in the presence of a specified reducing agent. The more focused independent claim additionally recites incubating recA1PI with 10 mM cysteine and separating recA1PI from the reducing agent.
Stated Advantages
Reduction or diminution of the yellow color associated with recA1PI when reducing agents are combined with chromatography.
Improved activity recovery.
Reduction of iron associated with recA1PI.
Documented Applications
Purification of cell culture derived human recombinant alpha1-protease inhibitor (recA1PI) from an aqueous solution.
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