High yield production and purification of recombinant staphylokinase for thrombolytic therapy.

Recombinant plasmids were constructed in which the signal sequence of the sak42D and the sakSTAR staphylokinase genes were replaced by an ATG start codon and which express staphylokinase under the control of a tac promoter and two Shine-Dalgarno sequences in tandem. Induction of transfected E. coli TGl cells in a bacterial fermentor produced intracellular staphylokinase representing 10 to 15% of total cell protein. Gram quantities of highly purified recombinant staphylokinase were obtained from cytosol fractions by chromatography, at room temperature, on SP-Sepharose and on phenyl-Sepharose columns, with yields of 50 to 70 percent. The material, at a dose of 4 mg/kg, did not produce acute reactions or affect body weight in mice. Intravenous administration of 10 mg SakSTAR over 30 minutes in five patients with acute myocardial infarction induced complete coronary artery recanalization, without associated fibrinogen degradation. However, neutralizing antibodies appeared in the plasma of all patients within 12 to 20 days. Thus, the present expression and purification method for recombinant staphylokinase yields large amounts of highly purified mature protein (approximately 200 mg per liter fermentation broth) suitable for a more detailed clinical investigation of its potential as a thrombolytic agent.

Ion-pair extraction method for quantitation of a bisquaternary ammonium compound, pancuronium bromide.

Pancuronium bromide (PCBr), a neuro-muscular blocking agent, was determined quantitatively in aqueous solution by ion-pair extraction. Separate determination of PCBr and degradation products was possible after thin layer chromatography. The procedure was developed by using a theoretical approach. Favorable conditions were calculated from the ion-pair extraction constant. The drug was determined with bromothymol blue at pH 9.0, using one extraction in the direct procedure and 2 successive extractions in the combined elution-extraction process after thin layer chromatography. In the direct method, 0.100 mg PCBr was determined with a reproducibility of +/- 1.0%.

Estimation of the amount of water removed by gap and atomization air streams during pelletization in a rotary processor.

The purpose of this study was to estimate the amount of water removed by the gap and atomization air streams during pelletization by the wet granulation technique in a rotary processor and to test the hypothesis that the influence of the water addition rate on the pellet size (1,7) mainly originates from differences in the amount of water removed by these air streams. Estimations from flow, temperature, and relative humidity measurements of the air streams, and estimations from moisture content measurements, indicated that approximately 3.6 g of water/min was removed during spheronization. The estimations from the air streams' characteristics obtained for the water loss during the water addition phase (2.6 g/min) reflected the water removal during the later stages and did not reflect the overall rate of water removal during the total water addition phase as estimated from moisture content measurements (2.0 g/min). This was attributed to the fact that estimations from the air streams' characteristics could not take into account the early stages of the water addition phase. In these early stages, the amount of water removed was very limited. Using the foregoing estimations, differences in amount of water removed by the air streams could be compensated by adding more water when the water addition rate was decreased. This resulted in similar pellet sizes (geometric mean diameter of 800-850 microns) using different water addition rates. Thus, the hypothesis that the major influence of the water addition rate on the pellet size mainly originates from differences in the amount of water removed by the gap and atomization air streams was confirmed.