Mitsui Toatsu Chemicals, Incorporated

Mitsuyoshi Morii

Masaharu Ohoka

Nobuhiro Kawashima

Toshihiko Suzuki

Noriko Morii

Katsuyuki Suzuki

Kunizo Mori

Kunizou Mori

Separation of tPA and undesired proteins is securely and effectivity accomplished by using a cation-exchanger in the two step procedure for selective elution of undesired proteins from the cation-exchanger on which tPA and undesired proteins are adsorbed: undesired proteins having the pI equivalent to or lower than that of tPA was eluted out in the first step and then undesired proteins having the pI equivalent to or higher than were eluted in the second step.

Method for purifying a crude tissue plasminogen activator preparation

Crude tPA containing, as an impurity, a protein which is reactive with an anti-human tPA antibody and has a molecular weight of 110,000.+-.20,000 daltons is purified by bringing the crude tPA into close contact with an affinity reagent containing an immobilized Kunitz inhibitor which is produced in seeds of Erythrina latissima and other Erythrina plants and acts as an inhibitor on trypsin, plasmin and tPA but does not act on urokinase, so that tPA is collected selectively.

Purificaiton procedure of tPA from crude preparations

Tissue plasminogen activator (tPA) species having a molecular weight of about 70,000 daltons is isolated in a purified form from a crude tPA preparation containing various tPA species having different molecular weights by bringing the crude tPA preparation into contact with a cation exchanger in advance and separating the desired tPA species selectively from the cation exchanger by means of the salt gradient elution method.

Process of purifying tPA

Process for separating single-chain TPA and double-chain from each other

A mixture containing a single-chain tissue plasminogen activator (tPA) and/or double-chain tPA is brought into close contact with a column carrying an immobilized Erythrina trypsin inhibitor as an affinity agent. Adsorbed protiens are eluted with eluents having different pHs with or without arginine or benzamidine, so that single-chain tPA is obtained in the eluent with a pH at least 4.5 and double-chain tPA is obtained in the eluent with a pH lower than 4.5.

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Yokohama, Japan

Mitsuyoshi Morii