Abstract: Objective: To explore the preparation process of lyophilization of recombinant IFN-α-2b-BCG(rBCG), and study the biological characteristics of lyophilized rBCG for clinical immunological activity to provide biological stability, safe and effective freeze-dried formulations.. Method: Vacuum-freeze-drying of recombinant IFN-α-2b-BCG, drawing the curve of lyophilization process, screening the best processing parameters. Comparing the number of viable cells with the plate count, before and after lyophilization, to calculate the survival rate. The acid-fast staining characteristics and morphology of the lyophilized rBCG was analyzed. Continuous measurement of the OD value of freeze-dried rBCG, drawing the growth curve. The plasmid stability of lyophilized rBCG was analysed by PCR and kanamycin-resistance. The level of IFN-α-2b expressed by rBCG was determined with ELISA. Results: The number of viable rBCG reached 6.5×106cfu/ml after lyophilization, the value was 1×107cfu/ml before freeze-drying, so survival rate was about 65%. The morphology, growth rate and acid-fast staining characteristics of lyophilized rBCG was not significantly different, from that of un-lyophilized. The stability rate of gene insertion（hIFN-α-2b）was 91.7%, loss rate 8.3%. The level of secretion of rIFN-α-2b by rBCG had no significant difference after lyophilization (p>0.05). Conclusion：Freeze-dried powder of hIFN-α-2b BCG was successfully prepared. After lyophilization, viable rBCG achieved 1×107cfu/ml, which is the standard of bio-immune-therapy needed. There was no significant difference in morphology, growth and expression of exogenous IFN-α-2b,comparing before with after lyophilization. Adverse effects of freezing, low temperature and vacuum drying of lyophilization process has on significant effects on hIFN-α-2b expression, plasmid insert, genetic stability of rBCG.

Key words: recombinant BCG, IFN-α-2b, bladder tumor, lyophilization