Method for making therapeutic enzyme compositions

Claim 1

In a method wherein a prothrombin complex-containing blood protein fraction is activated under conditions which produce enzymatically active blood clotting factors, the improvement comprising (a) selecting at least one of said proenzyme activating conditions which is to be varied to control the degree of activation; (b) prior to the completion of activation, determining the magnitude of the condition needed to activate the fraction to a predetermined degree of activation; (c) setting the condition to said magnitude; and (d) conducting the activation of the fraction in accordance with said condition; and (e) terminating the activation at said predetermined degree of activation.

Claim 8

The method of claims 4 or 6 wherein the degree of activation of the fraction is determined by assaying its nonactivated partial thromboplastin (NAPT) activity.

Claim 10

The method of claims 4 or 6 wherein the degree of activation of the fraction is determined by assaying its nonactivated partial thromboplastin (NAPT) activity and its factor VIII correctional activity.

Claim 20

A method of making an activated prothrombin complex which comprises (a) dissolving a paste of Cohn fraction IV-1; (b) adsorbing the clotting factors in the fraction onto calcium phosphate; (c) eluting the clotting factors from the calcium phosphate; (d) determining the nonactivated partial thromboplastin (NAPT) and factor VIII correctional times of the step (c) eluate; (e) where the nonactivated partial thromboplastin (NAPT) and factor VIII correctional times determined in step (d) are respectively greater than about 200 seconds and more than about 89 seconds, removing aliquots of the eluate and activating them for different periods of time; (f) assaying the nonactivated partial thromboplastin (NAPT) time for each aliquot; (g) determining the activation period which yields an nonactivated partial thromboplastin (NAPT) time of about from 70-100 seconds; (h) activating the remaining bulk of the step (c) eluate under substantially the same conditions as were used to activate the step (e) aliquots; (i) assaying the nonactivated partial thromboplastin (NAPT) time of the bulk at regular intervals before the end of the activation period determined in step (g); (j) terminating the activation of the bulk when the nonactivated partial thromboplastin (NAPT) time is about from 70 to 100 seconds; (k) concentrating the activated prothrombin complex by precipitation from polyethylene glycol (PEG); (l) dissolving the polyethylene glycol (PEG) precipitate in an aqueous solution containing about from 1 to 2 units of heparin/ml; and (m) lyophilizing the dissolved precipitate.

Claim 21

A method of making an activated prothrombin complex which comprises (a) dissolving a paste of Cohn fraction IV-1; (b) adsorbing the clotting factors in the fraction onto calcium phosphate; (c) eluting the clotting factors from the calcium phosphate; (d) determining the nonactivated partial thromboplastin (NAPT) and factor VIII correctional times of the step (c) eluate; (e) where the nonactivated partial thromboplastin (NAPT) and factor VIII correctional times determined in step (d) are respectively greater than about 200 seconds and more than about 89 seconds, removing aliquots of the eluate and activating them for different periods of time; (f) assaying the nonactivated partial thromboplastin (NAPT) time for each aliquot; (g) determining the activation period which yields an nonactivated partial thromboplastin (NAPT) time of about from 70-100 seconds; (h) activating the remaining bulk of the step (c) eluate under substantially the same conditions as were used to activate the step (e) aliquots; (i) assaying the nonactivated partial thromboplastin (NAPT) time of the bulk at regular intervals before the end of the activation period determined in step (g); (j) terminating the activation of the bulk when the nonactivated partial thromboplastin (NAPT) time is about from 70 to 100 seconds; (k) concentrating the activated prothrombin complex by precipitation from polyethylene glycol (PEG); (l) dissolving the polyethylene glycol (PEG) precipitate in an aqueous solution containing about from 1 to 2 units of heparin/ml; and (m) lyophilizing the dissolved precipitate.