Composition and method for preparing plasminogen
View Patent ↗Disclosed is both a method for preparing a plasminogen and a method for preparing a reversibly inactive acidified plasmin by activating the plasminogen. The prepared plasminogen is typically purified from a fraction obtained in the separation of immunoglobulin from Fraction II+III chromatographic process and eluted at a low pH. The prepared plasmin is isolated and stored with a low pH-buffering capacity agent to provide a substantially stable formulation. The reversibly inactive acidified plasmin may be used in the administration of a thrombolytic therapy.
1. A method for preparing plasminogen, the method comprising:
adding a buffer and a lysine derivative to a caprylate cake to obtain a plasminogen/lysine derivative-containing suspension, wherein the caprylate cake is a plasminogen-containing fraction resulting from caprylate precipitation of Cohn Fractions II+III;
extracting the suspension to obtain a plasminogen/lysine derivative-containing solution comprising the plasminogen;
adding the plasminogen/lysine derivative-containing solution to a cation-exchange resin to obtain a cation-exchange flow-through comprising the plasminogen;
adding the flow-through comprising the plasminogen to a plasminogen-specific absorbent material; and
eluting the plasminogen from the plasminogen-specific absorbent material to obtain a composition comprising the plasminogen.
2. The method of claim 1 , wherein the cation-exchange resin is a column.
3. The method of claim 1 , wherein the cation exchange resin and the plasminogen-specific absorbent material are equilibrated with the same buffer.
4. The method of claim 1 , wherein the cation exchange resin and the plasminogen-specific absorbent material are connected in series.
5. The method of claim 1 , wherein the lysine derivative is L-lysine or epsilon amino caproic acid (EACA).
6. The method of claim 5 , wherein the lysine derivative is L-lysine.
7. The method of claim 1 , wherein the step of extracting comprises extracting the suspension at a pH in a range of about 3.5 to about 10.5.
8. The method of claim 7 further comprising adding polyethylene glycol, metal oxide, ammonium sulfate, or a combination thereof to the plasminogen/lysine derivative-containing solution to precipitate impurities.
9. The method of claim 8 further comprising separating the precipitated impurities from the plasminogen/lysine derivative-containing solution prior to the step of adding said solution to the cation-exchange resin.
10. The method of claim 1 , wherein the step of eluting comprises eluting at a pH of about 3 to about 4.
11. The method of claim 1 further comprising removing or inactivating pathogens.
12. The method of claim 11 , wherein removing pathogens comprises inactivating viral pathogens and removing pathogens causing Transmissible Spongiform Encephalopathies (TSEs).
13. The method of claim 11 , wherein pathogens are removed or inactivated by a step selected from the group consisting of heat treatment, caprylate addition, solvent detergent addition, nanofiltration, and combinations thereof.
14. A method for preparing plasminogen, the method comprising:
adding a buffer and a lysine derivative to a caprylate cake to form a plasminogen/lysine derivative-containing suspension, wherein the caprylate cake is a plasminogen-containing fraction resulting from caprylate precipitation of Cohn Fractions II+III;
extracting the suspension at a pH of about 3.5 to about 10.5 to obtain a plasminogen/lysine derivative-containing solution comprising the plasminogen;
precipitating impurities from the plasminogen/lysine derivative-containing solution
adding the plasminogen/lysine derivative-containing solution to a cation-exchange resin to obtain a cation-exchange flow-through comprising the plasminogen;
adding the flow-through comprising the plasminogen to a plasminogen-specific absorbent material; and
eluting the plasminogen from the plasminogen-specific absorbent material to obtain a composition comprising the plasminogen.
15. A method for preparing a plasminogen, the method comprising:
precipitating a Cohn Fractions II+III comprising the plasminogen to obtain a caprylate cake I fraction (CCI), said CCI fraction comprising the plasminogen;
extracting the CCI fraction with a buffer and a lysine derivative to obtain a plasminogen/lysine derivative-containing solution;
adding the plasminogen/lysine derivative-containing solution to a cation-exchange resin to obtain a cation-exchange flow-through comprising the plasminogen;
adding the flow-through comprising the plasminogen to a plasminogen-specific absorbent material; and
eluting the plasminogen from the plasminogen-specific absorbent material to obtain a composition comprising the plasminogen.