复方益肝灵制剂中检出微生物的分型溯源与产品质量评价

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、RiboPrinter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 monitor microbial contamination and evaluate product quality of Yiganling oral tablets by microorganism identification and molecular ribotyping.Methods:The 138 Yiganling oral tablets/capsules in the national drug evaluation assay in the year 2014 were collected,and systematically exanimated by microbial limitation assay.Totally 36 microbial isolates from Yiganling oral tablets were collected,and analyzed by gram staining,microscope examination,biochemical identification,16S rDNA sequencing and molecular ribotyping for identification,ribotyping and tracing.Results:Biochemical analysis was reported as failure to identify microbial contaminants,while 16S rDNA sequencing and ribotyping analysis had consistent results that the contaminants were identified as Bacillus flexus.Dendrogram analysis by ribotyping showed that the contaminants can be divided into two subtypes,subtype I including 24 bacteria with homologous probability of more than 94% and subtype II including 12 bacteria with homologous probability of more than 99%.The homologous probability between the two subtypes was just 87.2%.The analysis of strain ribotyping results relative to product sampling batch showed that the distribution of each microbial subtype and the homologous relationship were concentrated and consistent with the production period and the production batch.Conclusion:Bacterium identification and typing results concluded that Yiganling oral tablets produced have high risk of microbial contamination,so microbial monitoring and timely check on contamination risks are intensively suggested in the process of production.The analysis of cases in this study proposed that establishment of molecular ribotyping methods in nonsterile drug products can effectively control products quality,and provide technical approach and data support for supervision and administration.