K 1K 2P u, a recombinant t-PA/u-PA Chimera with increased thrombolytic potency, consisting of amino acids 1 to 3 and 87 to 274 of human tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of human single chain urokinase-type plasminogen activator (scu-PA). Purification in centigram quantities and conditioning for use in man

Abstract

K 1K 2P u, a recombinant t-PA/u-PA chimera with increased thrombolytic potency in animal models of venous and arterial thrombosis, which consists of amino acids 1 to 3 and 87 to 274 of human tissue-type plasminogen activator (t-PA) and amino acids 138 to 411 of human single chain urokinase-type plasminogen activator (scu-PA), was produced and conditioned for use in patients. Chinese hamster ovary cells were transfected with an expression plasmid containing the K 1K 2P u cDNA, high producer cell lines were selected and scaled up in 800 cm 2 roller bottles, and 350 ml conditioned cell culture medium was harvested 3 to 7 times at 2 to 5 day intervals. Batches of 21 ± 4 liter (mean ± SD, n= 28) containing 1.8 ± 0.6 mg/l of K 1K 2P u related antigen were purified by chromatography on Copper chelate-Sepharose and immunoadsorption on an insolubilized murine monoclonal antibody (MA-IC8). Yields were 8.6 ± 3.4 mg K 1K 2P u per batch with a specific activity of 83,000 ± 44,000 IU/mg. The final material, obtained at a concentration of approximately 0.7 mg/ml, was dialyzed against 0.3 M NaCl, 0.02 M Tris-HC1 buffer, pH 7.5, containing 0.01% Tween 80 and 10 KIU/ml aprotinin. It was homogeneous on SDS-PAGE, contained 6.5 ± 6.9 percent two chain material and the contamination with murine monoclonal antibody was less than 0.1 percent. After filtration of pools of 3 to 5 selected batches on 0.22 μm Millipore filters the material was sterile and virus free by routine screening; it was obtained at a concentration of approximately 0.5 mg/ml with a specific activity of 110,000 ± 16,000 IU/mg (mean ± SD, n= 3) and an endotoxin content of 0.5 to 7 units/mg. Bolus injection at a dose of 1 mg/kg in mice did not produce weight loss within 8 days. Thus, this material appears to be suitable for the investigation on a pilot scale of the pharmacokinetic and thrombolytic properties of K 1K 2P u in patients with thromboembolic disease.