Stabilization of a freeze-dried recombinant streptokinase formulation without serum albumin.

AIM: To evaluate the stability and content homogeneity of a new freeze-dried and albumin-free formulation of recombinant streptokinase (SKr) that has recently been approved by the Cuban National Center for the Quality Control of Medicaments. MATERIALS AND METHODS: The new formulation was stored at the intended recommended storage temperature of 4 degrees C, and under accelerated storage conditions (37 degrees C). The stability of the product was also examined after reconstitution and storage at room temperature (28 degrees C) for 24 h. Samples were periodically subjected to biological activity assays (S-2251 chromogenic-substrate method or in vitro clot-lysis assay), sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/PAGE), pyrogen and sterility testing, abnormal toxicity screening, organoleptic evaluation, and measurement of residual moisture and pH. RESULTS AND DISCUSSION: Accelerated storage (37 degrees C) data showed biochemical stability of SKr throughout the 6-month study with activity, remaining between 90 and 110% of its nominal value (0.75 x 10(6) IU/mL). SDS/PAGE-determined purity showed that SKr remained above 97 %. Furthermore, the formulation was non-pyrogenic, non-toxic, sterile and organoleptically acceptable. Real-time storage data confirmed the excellent biochemical long-term (30 months) stability of the new formulation of SKr. Comparison with other freeze-dried preparations showed that the new formulation was organoleptically better. The formulation was stable after reconstitution and storage at 28 degrees C for 24 h. The content homogeneity of this new formulation was also satisfactory. CONCLUSIONS: This study demonstrated the stability and the content homogeneity of this formulation, despite the absence of albumin as stabilizer.

Immunoaffinity purification of recombinant hepatitis B surface antigen from yeast using a monoclonal antibody.

A murine monoclonal antibody developed for the purification of recombinant hepatitis B surface antigen was immobilized on a chromatographic support and used to adsorb and purify the recombinant antigen from yeast. The adsorption-elution behaviour was first investigated using monoclonal antibody-coated enzyme-linked immunosorbent assay plates and performing adsorption, washing and elution procedures with different elution agents. It was found that 3 M KSCN and 8 M urea at neutral pH disrupted antigen-antibody interactions in both systems. The procedure for washing the immunoaffinity column was optimized, using different salts and detergents. The best results were obtained by applying the starting material in 1 M NaCl and washing with the same buffer. The use of 0.1% sodium deoxycholate in the washing buffer reduced about 20-fold lipopolysaccharide contamination in the eluates as compared with washing without detergent. The relationship between bed height and the adsorption capacity of the column was studied, and it was found that the dynamic capacity decreased twice on reducing its length/diameter ratio tenfold. The recovery of antigen was not affected by increasing the flow-rate up to 25 cm/h but decreased at higher values. Using the optimum conditions, the affinity column was able to purify the recombinant hepatitis B surface antigen to more than 90% purity and a 65% antigen recovery was obtained.

[Research of antifungal substances secreted by higher fungi in culture]. / Recherche de substances antifongiques sécrétées par les champignons supérieurs en culture.

A screening in vitro of antifungic activity of 24 strains of Basidiomycetes was realized with their culture filtrate. Lycoperdon perlatum Pers. = Pers., Oudemansiella platyphylla (Pers. ex Fr.) Mos., Agrocybe dura (Bolt) Singer have shown an activity against Candida albicans, Candida tropicalis and Aspergillus fumigatus; Pholiota spumosa (Fr.) Singer towards Botrytis cinerea and Lycoperdon perlatum Pers. = Pers. towards Alternaria solani, Botrytis cinerea and Verticillium dahliae. More extensive studies of the kinetics of antifungic substances production from Lycoperdon perlatum Pers. = Pers. were effectued with 2 different media and 2 different seeding technics on the strains which were sensible to the inhibitory character of this Gasteromycete.