Method for removing a lytic enzyme from a heterogeneous mixture

Patent metadata

Field	Value
Publication number	US-9328338-B2
Application number	US-201213532997-A
Country	US
Kind code	B2
Filing date	Jun 26, 2012
Priority date	Jun 30, 2011
Publication date	May 3, 2016
Grant date	May 3, 2016

Abstract

The invention relates to purification of an intact, non-degraded macromolecule from a biological mixture comprising the macromolecule in the presence of its lytic enzyme. The method comprises providing the biological mixture as a heterogeneous mixture comprising the lytic enzyme, at least partially, in soluble form and the macromolecule, at least partially, in non-soluble form; batch-wise contacting the heterogeneous mixture with an immobilized inhibitor of the lytic enzyme; increasing the solubility of the macromolecule in the mixture; and removing the immobilized inhibitor from the mixture.

First claim

What is claimed is:

1. A method for removing a lytic enzyme from a precipitated biological mixture, the biological mixture comprising the lytic enzyme capable of degrading fibrinogen, fibrinogen and aluminum hydroxide particles, the method comprising the steps of:
providing the precipitated biological mixture;
providing an inhibitor of the lytic enzyme immobilized on a carrier;
partially dissolving the precipitated biological mixture with an aqueous solution under conditions that allow the lytic enzyme to dissolve to obtain a heterogeneous mixture comprising dissolved lytic enzyme and solid particles including fibrinogen and aluminum hydroxide particles;
contacting the heterogeneous mixture with the immobilized inhibitor in batch form followed by a step of fully dissolving the fibrinogen solid particles in the mixture thereby obtaining a mixture including immobilized inhibitor, aluminum hydroxide particles and fully dissolved fibrinogen; and
separating the aluminum hydroxide and the immobilized inhibitor and the lytic enzyme bound to the inhibitor from the fully dissolved fibrinogen by centrifugation and/or filtration, wherein the partially dissolving conditions are selected from the group consisting of a pH range of 7.2-7.3, a temperature range of 30-32C, an ethanol concentration in the range of 0.2 to 5%, and a combination thereof.

2. The method according to claim 1 , wherein the partially dissolving conditions comprise a pH range of 7.2-7.3, a temperature range of 30-32° C., and an ethanol concentration in the range of 0.2 to 5%.

3. The method according to claim 1 , wherein the precipitated biological mixture derives from a blood fraction.

4. The method according to claim 1 , wherein the precipitated biological mixture is a by-product precipitate from the manufacture process of factor VIII.

5. The method according to claim 1 , wherein the lytic enzyme is a protease.

6. The method according to claim 1 , wherein the inhibitor is an amino acid analog.

7. The method according to claim 1 wherein the macromolecule is fibrinogen, the lytic enzyme is plasmin and/or plasminogen, and the inhibitor is a lysine analog.

8. The method according to claim 7 , wherein the lysine analog is tranexamic acid.

9. The method according to claim 1 , wherein the precipitated biological mixture is provided frozen.

10. The method according to claim 9 , further comprising the step of reducing the mean particle size of the frozen precipitated biological mixture to about 2-8 mm.

11. The method according to claim 10 , wherein the reduction in the mean particle size is carried out mechanically.

12. The method according to claim 11 , wherein the reduction in the mean particle size is carried out using a blender machine.

13. The method according to claim 1 , wherein the contacting step is carried out for more than 30 minutes.

14. The method according to claim 1 , wherein the separating step is carried out by centrifugation and/or filtration.

15. The method according to claim 1 , wherein the steps of partially dissolving the precipitated biological mixture and contacting the heterogeneous mixture with the immobilized inhibitor are carried out simultaneously.

Assignees

• Meidler Roberto
• Raver-Shapira Nina
• Bar Liliana
• Belyaev Oleg
• Nur Israel
• Omrix Biopharmaceuticals Ltd

Inventors

• Meidler Roberto
• Raver-Shapira Nina
• Bar Liliana
• Belyaev Oleg
• Nur Israel

Classifications

• C12Y304/21007

  Plasmin (3.4.21.7), i.e. fibrinolysin · CPC title

• C07K14/811

  Serine protease (E.C. 3.4.21) inhibitors · CPC title

• C07K14/81

  Protease inhibitors · CPC title

• C12N9/6435

  Plasmin (3.4.21.7), i.e. fibrinolysin · CPC title

• C07K1/22 Primary

  Affinity chromatography or related techniques based upon selective absorption processes · CPC title