Process for plasminogen purification starting from human plasma
Inventors
Ascione, Ester • Farina, Claudio • Lazzarotti, Alessandra • MADDALUNO, Marcella • Nardini, Claudia
Assignees
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Publication Number
US-11591587-B2
Publication Date
2023-02-28
Expiration Date
Abstract
The present invention provides a process for the production of human plasminogen starting from human plasma or a fractionation intermediate thereof. The main stages of the process are: a step of virus inactivation, in which human plasma is contacted with a solvent/detergent mixture, a single affinity chromatographic step and a virus removal nanofiltration step. This process is scalable up to industrial level and it provides, without adding any protease inhibitor, a functional and intact finished product suitable to be administered for the treatment of human diseases due to genetic plasminogen deficiency.
Core Innovation
The document describes an industrial scale process for obtaining a highly purified and virus safe plasminogen that contains more than 98% wt native form plasminogen (Glu-Pg) suitable to be administered to humans for therapeutic purposes. The process starts from frozen human plasma and includes solvent/detergent viral inactivation, while employing no protease inhibitor during the process.
A central part of the process is a single affinity chromatographic step on L-lysine immobilized cross-linked agarose resin. The affinity chromatography resin is ECH-Lysine SEPHAROSE® 4 Fast Flow, and the document specifies sodium phosphate and ε-aminocaproic acid together with sodium chloride at pH 7.4 for plasminogen elution to obtain a native form plasminogen eluate.
The document further defines downstream virus removal by nanofiltering the native form plasminogen eluate through a nanofilter to remove viral contaminants. In a sequenced embodiment, it also includes filtration steps such as a 0.22 μm filter for particulate removal, dialysis membrane filtration with a 0.1M NaCl solution to remove ε-aminocaproic acid, and nanofiltering through a 20 nm filter.
Claims Coverage
Two independent claims are provided (clm-00001 and clm-00007). Across these, the inventive features cover a virus safe industrial process producing >98% wt Glu-Pg from frozen human plasma without protease inhibitor, combining solvent/detergent inactivation, a single specified L-lysine affinity chromatography step, and nanofiltration, with a sequenced embodiment that further specifies filtration and conditioning steps.
Industrial scale production of >98% wt Glu-Pg from frozen human plasma without protease inhibitor
An industrial scale process starting from frozen human plasma to obtain a highly purified and virus safe plasminogen containing more than 98% wt native form plasminogen (Glu-Pg) suitable to be administered to humans for therapeutic purposes, wherein no protease inhibitor is employed during the process.
Solvent/detergent viral inactivation for virus safe Pg
Contacting the frozen human plasma with a solvent/detergent mixture for virus inactivation as part of the industrial scale process.
Single L-lysine affinity chromatography on ECH-Lysine SEPHAROSE 4 Fast Flow with defined buffer system
Subjecting the human plasma after virus inactivation to a single affinity chromatographic step on L-lysine immobilized cross-linked agarose resin where the affinity chromatography resin is ECH-Lysine SEPHAROSE® 4 Fast Flow, using sodium phosphate, ε-aminocaproic acid, and sodium chloride at pH 7.4 to obtain a native form plasminogen eluate.
Nanofiltration to remove viral contaminants producing virus safe Pg with >98% wt Glu-Pg
Nanofiltering the native form plasminogen eluate from the affinity chromatographic step through a nanofilter to remove viral contaminants, wherein the filtrate comprises purified and virus safe plasminogen containing more than 98% wt native form plasminogen (Glu-Pg) suitable to be administered to humans for therapeutic purposes.
Sequenced filtration and virus inactivation train to reach >98% wt Glu-Pg without protease inhibitor
An industrial scale process in sequence including filtration of human plasma, contacting with a solvent/detergent mixture to inactivate viruses, adding castor oil, filtering through a depth filter, performing a single affinity chromatography using ECH-Lysine SEPHAROSE® 4 Fast Flow, filtering through a 0.22 μm filter for particulate removal, dialysis membrane filtration to remove ε-aminocaproic acid, filtering through a 0.1 μm filter, and nanofiltering through a 20 nm filter to obtain pure and virus safe plasminogen containing more than 98% wt native form plasminogen (Glu-Pg), wherein no protease inhibitor is employed during the process.
The independent claims focus on an industrial process that yields virus safe plasminogen enriched to more than 98% wt native form (Glu-Pg) from frozen human plasma, using solvent/detergent viral inactivation, a single defined L-lysine affinity chromatography on ECH-Lysine SEPHAROSE® 4 Fast Flow, and nanofiltration, with an embodiment that additionally specifies castor oil addition, multiple filtration steps, dialysis for ε-aminocaproic acid removal, and a 20 nm nanofilter, all while employing no protease inhibitor.
Stated Advantages
Produces purified and virus safe plasminogen containing more than 98% wt native form plasminogen (Glu-Pg) suitable to be administered to humans for therapeutic purposes.
Achieves virus safety by removing viral contaminants through nanofiltration.
Operates without employing a protease inhibitor during the process.
Documented Applications
Plasminogen suitable to be administered to humans for therapeutic purposes.
Industrial scale process for obtaining highly purified and virus safe plasminogen.
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