Affinity fractionation of heparin on immobilized Concanavalin A

Claim 1

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.

Claim 2

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.

Claim 14

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.

Claim 15

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.