Urokinase (peptidolytic)

White or almost white, amorphous powder.

Urine collection, precipitation, acidification by adsorption method Collect male urine in a special plastic bucket. The conductivity of the urine is equivalent to 20-30m·Ω-1, the Ph is below 6.5, the number of bacteria is below 1000/ml, and 0.8% phenol is added for preservation in summer. Urine must be processed within 8h. Cool the freshly treated urine to below 1010C, adjust Ph to 8.5 with sodium hydroxide (3nol/L), let stand for 1h, siphon the supernatant, and neutralize OH-1 to Ph5 with hydrochloric acid (3nol/L) 5.5. Get acidified urine. Fresh male urine NaOH, Ph8 → precipitated supernatant urine HCl, Ph5 → acidified acidified urine adsorption and elution Take acidified urine, add 10g diatomaceous earth (1%) per liter of urine, at a temperature below 50C Continue stirring to make it adsorb for 1h. The adsorbate is washed with cold water of about 50, then packed into a column (column ratio 1:1), washed with ammonia water (0.02%) until the outflow solution becomes clear, and then use ammonia water (0.02%) containing sodium chloride (1mol/L) The urokinase is eluted until the collection starts when the eluate becomes mixed. The collection volume is about 15L of eluate (100U/ml, 3000U/mg) per ton of urine. Acidify urine diatomaceous earth→adsorb cold water 50^→wash diatomaceous earth column NH3^·H2O→wash eluent to remove pyrogen, pigment eluent adjust Ph8 with saturated sodium dihydrogen phosphate, adjust conductivity with sodium chloride Pass through a QAE-Sephadex column at 22mm·Ω-1 (this column is pre-equilibrated with pH8r phosphate buffer), and collect the effluent. The chromatographic column should be washed continuously with phosphate buffer (retention volume not less than 3 times the column volume) and combined with the effluent. Eluent QAE-Sephadex column → depyrogenation, pigment effluent, CMC concentrated effluent, adjusted Ph4.2 with acetic acid (1mol/L), adjusted with distilled water, the conductivity is equivalent to 16-17mΩ-1, and passed the CMC column (the column Slowly the solution with 0.1mol/L, Ph4.2 acetic acid in advance, the conductivity is equivalent to 17mΩ-1, equilibrate for about 12h) The chromatographic column is continued to be washed with not less than 10 times the column volume of Ph4.2 acetic acid-sodium acetate buffer solution Column, after the elution is completed, use ammonia (0.1%) with sodium chloride (0.1mol/L), Ph11.5～11.8 to elute the urokinase, and the urokinase eluate flows out into filaments, and some of them are collected and eluted. Liquid (3000～40, 000U/ml, 15000～20000U/mg). Effluent CMC column→Concentration}CMC column NH3·H2, Ph11.5→Wash the eluent, dialyze, freeze-dry the eluent is dialyzed in 40 pairs of water for 24h, change the water 3 to 4 times, and centrifuge the dialysate to remove precipitation , Freeze-drying, that is urokinase. Eluent water 40;24h^→dialysis, freeze-dried product sterilization, sub-packaging After the urokinase is qualified for sampling, it is diluted, sterilized, added with excipients, sub-packed, and lyophilized to obtain a urokinase preparation. Urokinase excipient→sterilized, freeze-dried urokinase preparation

Urokinase is a protease that cleaves plasminogen to form plasmin. It binds to cell surface receptors (uPAR), regulates cell adhesion tissue remodeling and migration, and also activates intracellular signal transduction pathways. Urokinase is composed of three domains: the amino terminal domain homologous to epidermal growth factor, which is a protease domain bound to the carboxy terminus through the central kringle domain.