US 4,539,398 A · Affinity fractionation of heparin on immobilized Concanavalin A

Abstract

A highly discriminating technique utilizing Concanavalin A, immobilized on a solid substrate, e.g. sepharose 4B, for the fractionation of heparin is disclosed. The heparin to be fractionated is incubated with antithrombin whereby a fraction complete with the antithrombin and the whole is then either passed through a column of the immobilized Concanavalin A or slurried with same. In both modes that fraction of the heparin complexed with the antithrombin is selectively absorbed by the immobilized Concanavalin A and constitutes the anticoagulant active fraction. That heparin which complexes with the antithrombin can be further fractionated to yield yet more highly active fractions by complexing the heparin in stages, adding an increment of a molar amount of antithrombin in each stage. A high molecular weight fraction (18,000-22,000 daltons) or a low molecule weight fraction (6,000-8,000 daltons), isolated from the heparin of animal tissue origin by chromatography, are the preferred heparin preparations for complexing with the antithrombin.

Claims23

Dependent claims

A method for affinity fractionation of heparin and isolation of a highly active anticoagulant fraction, said process comprising:

(a) providing Concanavalin A immobilized on a solid substrate;
(b) providing a heparin preparation of animal tissue origin;
(c) incubating said heparin preparation with a quantity of antithrombin extracted from mammalian plasma to complex a portion of the heparin with the antithrombin;
(d) contacting the incubation product of step c with said immobilized Concanavalin A to bind said complex to the immobilized concanavalin A solid phase and to separate a depleted heparin pool as the liquid phase;
and
(e) selectively eluting the heparin from the solid phase and recovering the eluted heparin as said highly active anticoagulant fraction.

3 more elements

A method for affinity fractionation of heparin from a heparin pool containing heparin capable of binding with antithrombin to separate the antithrombin binding heparin into fractions of varying anticoagulant activity, said process comprising:

(a) providing a heterogeneous heparin pool of animal tissue origin, said pool containing heparin of varying anticoagulant activity;
(b) incubating said heparin of step a with a quantity of antithrombin being sufficient only to complex a fraction of the heparin constituting a minor portion of the heparin in the pool capable of binding with antithrombin, said fraction of heparin having a higher antithrombin binding affinity than that of the heparin which remains in the uncomplexed pool, to produce a heparin-antithrombin complex in which the complexed heparin has a higher specific anticoagulant activity than the heparin remaining in the depleted pool;
(c) isolating the complex of step b from the heparin pool;
(d) separating the heparin from the antithrombin-heparin complex isolated in step c to produce heparin of higher specific anticoagulant activity than the specific activity of the depleted heparin pool;
(e) incubating said depleted heparin pool with a larger molar ratio of antithrombin than used in step b to form an antithrombin-heparin complex in which the heparin of the complex has a lower specific anticoagulant activity than the heparin product of step d but higher than the specific activity of the heparin remaining in the now further depleted heparin pool;
(f) isolating the complex of step e from the now further depleted heparin pool;
and,
(g) separating the heparin from the antithrombin-heparin complex isolated in step f to produce heparin of lower specific anticoagulant activity than the specific activity of the heparin produced in step d and higher than the specific activity of the heparin remaining in the further depleted heparin pool.

5 more elements

Dependencies are inferred from the claim text.

Affinity fractionation of heparin on immobilized Concanavalin A

Life of this patent

Affinity fractionation of heparin on immobilized Concanavalin A

Life of this patent

Abstract

Claims23