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目的:对复方益肝灵制剂中检出的微生物进行鉴定与分型,评估产品微生物污染的风险,评价产品质量。方法:收集整理2014年国家药品评价性抽验中,复方益肝灵制剂共计138件,经微生物限度方法学验证,对制剂中检出的微生物共36株,利用革兰染色、镜检、生化鉴定、16S rDNA测序、Ribo Printer核糖体分型等方法,进行鉴定、分型与溯源。结果:生化方法无法有效鉴定该微生物污染物;核酸测序与核糖体分型的鉴定结果一致,均为弯曲芽孢杆菌(Bacillus flexus)。核糖体分型的类聚分析结果表明:微生物污染物可分为2个亚型,其中亚型Ⅰ包含24个菌株,菌株间同源性关系>94%,亚型Ⅱ包含12个菌株,菌株间同源性关系>99%,2个亚型之间的同源性关系仅为87.2%;菌株分型结果与产品抽样批号的比较中发现,微生物各亚型的分布、同源性关系与产品的生产时段、生产批号呈现集中性和一致性的特点。结论:该制剂生产工艺流程中存在微生物污染的风险,应在生产过程中进行微生物监控,及时排查污染隐患。本研究通过上述案例分析,拟建立非无菌制剂中微生物的鉴定与分型方法及微生物污染物调查和追溯解决方案,为非无菌制剂的质量控制、监督抽验和用药风险评估提供技术手段和数据支持。
OBJECTIVE: To identify and classify the microorganisms detected in compound Yiganling agents, assess the risk of microbial contamination and evaluate the quality of products. Methods: In 2014, a total of 138 samples of Yigangan were collected and evaluated. According to the microbial limit test, 36 strains of microorganisms were detected in the preparations. Gram stain, microscopic examination and biochemical identification , 16S rDNA sequencing, Ribo Printer ribotyping methods, identification, typing and traceability. Results: The biochemical method could not effectively identify the microbial contaminants. The results of nucleic acid sequencing and ribotyping were the same, which were all Bacillus flexus. The results of cluster analysis showed that there were two subtypes of microbial contaminants, of which 24 strains were subtype Ⅰ, the homology was> 94% among isolates, 12 strains were subtype Ⅱ, Homology relationship> 99%, homology between the two subtypes was only 87.2%; comparison of strain typing results and product sampling lot found that the distribution and homology of each subtype of microorganism Product production period, the production batch number showed the characteristics of concentration and consistency. Conclusion: There is a risk of microbial contamination in the production process of the preparation. Microbiological monitoring should be carried out in the production process, and pollution risks should be promptly investigated. Through the above case analysis, this study intends to establish the identification and typing methods of microorganisms in non-sterile preparations and to investigate and trace the solution of microbial contaminants so as to provide technical measures for the quality control, supervision sampling and drug risk assessment of non-sterile preparations, data support.