K1K2Pu, 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.
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.
Collen
D
D
Center for Thrombosis and Vascular Research, University of Leuven, Belgium.
Nelles
L
L
De Cock
F
F
Lemmens
G
G
Van Coetsem
T
T
Demarsin
E
E
Lijnen
H R
HR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA / Q000235:genetics; D005343:Fibrinolytic Agents / Q000302:isolation & purification* / Q000494:pharmacology / Q000633:toxicity; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D010960:Plasminogen Activators; D016133:Polymerase Chain Reaction; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification* / Q000494:pharmacology / Q000633:toxicity; D015912:Thrombolytic Therapy; D010959:Tissue Plasminogen Activator / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology / Q000633:toxicity; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology / Q000633:toxicity
D005343:Fibrinolytic Agents; C085011:K1K2Pu; D011993:Recombinant Fusion Proteins; D004247:DNA; D010960:Plasminogen Activators; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator