宿主细胞残留DNA片段大小分布检测方法的建立及验证

Development and validation of a method for detection of size distribution of residual host cell DNA fragments

目的建立宿主细胞残留DNA片段大小分布的检测方法,并进行验证,为监控疫苗生产过程提供依据。方法基于高灵敏的微型化芯片毛细管电泳法,用MDCK细胞基因组DNA作为阳性对照品,建立检测方法,并验证方法的检测范围、准确性及精密度。利用建立的方法分析MDCK细胞基质流感疫苗生产过程中间品(H1N1型流感病毒收获液、病毒微滤液、核酸酶处理收获液、浓缩病毒液、层析病毒液)残留DNA片段大小分布。结果 MDCK细胞基因组浓度为933.06 ng/μL,A_(260/280)值为2.079,可作为阳性对照。该方法检测范围为35~10 380 bp,精密度检测CV均小于10%。用该方法检测H1N1型流感病毒疫苗中间品,结果显示宿主细胞基因组大片段被核酸酶处理成小片段,层析病毒液300 bp以下片段占总外源DNA的84%。结论建立了一种测定MDCK细胞基质流感疫苗宿主细胞残留DNA片段大小分布的检测方法,该方法精密度良好,有望用于连续细胞系衍生生物制品的原液检定和质量控制。

Objective To develop and validate a method for detection of size distribution of residual host cell DNA fragments,and to provide an evidence for monitoring the vaccine production process. Methods Based on a highly sensitive miniaturized chip-based capillary electrophoresis,intermediate products in the production of MDCK cells-based influenza vaccine were detected by using the MDCK genomic DNA as a positive control. The developed method was verified for detection range,accuracy and precision,and used for detection of size distribution of residual host cell DNA fragments in the intermediate products(harvest of influenza H1N1 virus,microfiltration fluid of virus,virus digested with nuclease,concentrated virus and the virus purified by chromatography). Results The concentration of genome of MDCK cells was 933. 06 ng/μL,while the A_(260/280) value was 2. 079,indicating that the genome could be used as positive control. The detection range of the developed method was 35 ~ 10 380 bp,while the CVs of test results for precision were less than 10%. The detection results of intermediate products of influenza H1N1 virus vaccine by the method showed that the large fragments of host cell genome were digested into small fragments with nuclease,and the fragments at lengths of less than 300 bp accounted for 84% of total exogenous DNA of virus purified by chromatography. Conclusion A method for detection of the size distribution of residual host cell DNA fragments in MDCK cells-based influenza vaccine was developed,which showed high precision,and was expected to be used for the quality control of continuous cell linederived biological products.