K1K2Pu, 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
K1K2Pu cDNA, high producer cell lines were selected and scaled up in 800 cm2 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
K1K2Pu related
antigen were purified by chromatography on
Copper chelate-
Sepharose and immunoadsorption on an insolubilized murine
monoclonal antibody (MA-1C8). Yields were 8.6 +/- 3.4 mg
K1K2Pu 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-HCl
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 microns 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
K1K2Pu in patients with thromboembolic disease.