Immuno Aktiengesellschaft

Baxter Aktiengesellschaft

Immuno Aktiengesellschaft Fur Chemisch-medizinische Produkte

Ernst Hetzl

Yendra Linnau

Hans-Peter Schwarz

Johann J Eibl

Wolfgang Sch&ouml;nhofer

H Peter Matthiessen

Silvia Neppl

A method of purifying antithrombin III (AT III) from a starting material containing an AT III/heparin complex or an AT III/heparinoid complex is disclosed. First, the method comprises adsorbing the AT III/heparin complex or the AT III/heparinoid complex on an anion exchanger material. Second, the method involves separating the AT III from the adsorbed AT III/heparin complex or an AT III/heparinoid complex by elution with a buffer having a pH ranging from 8.5 to 10.5 and a conductivity between 10 and 60 mS.

Method for purification of antithrombin III using an anion exchanger

A process for preparing lys-plasminogen from blood plasma or blood plasma products having a specific activity of at least 17.5 caseinolytic units/mg protein and at lease 50 .mu.moles/g protein nitrogen as well as a an electrophoretic purity of at least 90% is presented. Cohn fraction III is obtained by phosphate precipitation from blood plasma or blood plasma products. A crude plasminogen fraction is then obtained by precipitating most non-plasminogen proteins with ethanol. The crude plasminogen is absorbed on immobilized lysine affinity column. Elution followed by precipitation obtains a purified plasminogen. The purified plasminogen is converted to lys-plasminogen with plasmin having an activity from 0.01 to 0.1 .mu.moles/ml min relative to chromogenic substrate H-D-Val-L-Leu-L-Lys-p-nitroanilide by dialyzing at a temperature of about 1.degree. to 20.degree. C. for a time period of about 6 to 60 hours. The conversion is interrupted by adding aprotinin. The resulting lys-plasminogen is lyophilized to produce a lyophilized lys-plasminogen.

Method of producing LYS-plasminogen

To produce lys-plasminogen having a specific activity of at least 17.5 caseinolytic units/mg protein and at least 50 mymoles/g protein nitrogen as well as an electrophoretic purity of at least 90%, plasminogen from plasma, a plasminogen-containing fraction or a tissue culture is adsorbed on immobilized lysine for the purpose of purification, is eluted and is recovered from the eluate by a protein precipitating agent. A solution of the thus purified plasminogen is adjusted to a plasmin activity ranging between 0.005 and 0.2 mymoles/ml min relative to chromogenic substrate H-D-valyl-L-leucyl-L-lysine-p-nitroanilide dihydrochloride, is maintained at a temperature of from +1.degree. C. to +20.degree. C. for a period of from 6 to 60 hours in order to provoke an enzymatic-proteolytic conversion of plasminogen into lys-plasminogen, whereupon the enzymatic action is interrupted and lys-plasminogen is isolated.

Method of producing lys-plasminogen

In a method of producing an antithrombin III-heparin or an antithrombin III-heparinoid concentrate, heparin or heparinoid is added to human plasma or to antithrombin III containing plasma fractions to form an antithrombin III-heparin or antithrombin III-heparinoid complex, the complex thus formed is adsorbed on an anion exchanger, and eluted with a salt solution; then the antithrombin III-heparin or antithrombin III-heparinoid complex contained in the eluate is separated from salts and undesired proteins, and the resulting product is loyphilized.

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Vienna, Austria

Ernst Hetzl