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目的研究不同用量卵磷脂中和剂和PB缓冲液,在双价肾综合征出血热灭活疫苗(Vero细胞)无菌检查中的应用。方法取双价肾综合征出血热灭活疫苗(Vero细胞)转移至卵磷脂中和剂中(摇匀后放置5 min),按薄膜过滤法过滤后,用0.15 mol/L PB缓冲液(30~35℃保温)冲洗滤膜,加入液体培养基培养,进行不同用量卵磷脂中和剂和PB缓冲液适用性试验。用选定的试验条件进行无菌检查方法的适用性试验(薄膜过滤法)及阳性对照验证试验。结果采用10 ml卵磷脂中和剂中和10瓶供试品(摇匀后放置5 min),用0.15 mol/L PB缓冲液(30~35℃保温)冲洗滤膜,每张滤膜每次冲洗量为100 ml,共冲洗2次(末次冲洗液中含小于100 cfu的菌液),该方法过滤速度快,卵磷脂冲洗完全,无残留、不影响结果判定,确定适用。无菌检查方法的适用性试验中,供试品各滤筒中的试验菌与阳性对照比较均生长良好,达到《中国药典》三部(2015版)要求,应用于双价肾综合征出血热灭活疫苗(Vero细胞)的无菌检查中无抑菌作用或其抑菌作用可忽略不计。以金黄葡萄球菌和黑曲霉作为阳性对照菌,0和17 d培养到期后加入阳性菌,对比试验显示试验菌均生长良好。结论该方法适用于双价肾综合征出血热灭活疫苗(Vero细胞)无菌检查。
Objective To study the application of different dosage of lecithin neutralizer and PB buffer in the sterility test of Vero cells inactivated by divalent renal syndrome (VER). Methods Vero cells were transferred into lecithin neutralizer (shaking for 5 min) and filtered by membrane filtration. The cells were treated with 0.15 mol / L PB buffer (30 ~ 35 ℃ insulation) rinse the filter, added to the liquid culture medium, the amount of lecithin neutralizer and PB buffer applicability of the test. Suitability test (membrane filtration method) and positive control verification test of sterility test method with selected test conditions. Results Ten bottles of the test sample were neutralized with 10 ml of lecithin neutralizer (shaken for 5 min) and washed with 0.15 mol / L PB buffer (30-35 ° C) Rinse volume of 100 ml, a total of 2 rinse (the last wash containing less than 100 cfu bacteria), the method of filtration speed, lecithin rinse completely, no residue, does not affect the outcome of the decision to determine the applicable. In the applicability test of the aseptic test method, the test bacteria in each filter cartridge of the test product all grew well compared with the positive control and reached the requirement of “Chinese Pharmacopoeia” (2015 edition) Vaccines (Vero cells) have no bacteriostatic effect or negligible antibacterial activity in their sterility test. Staphylococcus aureus and Aspergillus niger as positive control bacteria, 0 and 17 d after the expiration of culture added positive bacteria, the comparative test showed that the test bacteria grew well. Conclusion This method is suitable for the sterility examination of Vero cells inactivated by dengue fever.