CHO宿主细胞DNA残留量检测方法的建立

目的建立检测生物制品中CHO宿主细胞DNA残留量的方法,制备检测CHO细胞DNA残留量的内部参考品,为国家相关标准的制定和修订提供依据。方法分别采用酚:氯仿:异戊醇抽提、DNA提取试剂盒和高盐抽提3种方法提取CHO细胞DNA,比较3种方法的提取效果;用地高辛标记最佳方法提取的CHO细胞DNA探针,并优化标记体系,直接法检测探针的标记效率;采用3种方法处理CHO细胞DNA内部参考品(1组不作处理;2组加入牛血清白蛋白和蛋白酶K;3组按2组方法处理后,增加DNA抽提步骤),通过点杂交法,用标记的探针检测上述样品的灵敏度。结果高盐抽提法提取的CHO细胞DNA效果较好;体系4(DNA10μg,Vial416μl,总体积64μl)标记效率最高;经不同方法处理的1、2、3组CHO细胞DNA内部参考品的检测灵敏度分别为1pg、10pg和1ng。结论建立的地高辛标记CHO细胞DNA探针-点杂交检测CHO细胞DNA残留量的方法稳定可靠,可用于生物制品中CHO宿主细胞DNA残留量的检测。

Development of A Method for Determination of Residual DNA in CHO Host Cells

Objective To develop a method and prepare an internal reference for determination of residual DNA in CHO host cells in biologics,and provide a basis for the establishment and revision of the relevant national standards.Methods DNAs were extracted from CHO cells with phenol:chloroform:isopentanol,DNA extraction kit and high salt method respectively,and the extraction effects were compared.The DNA probe extracted by the optimal method was labeled with digoxin,based on which the volumes for labeling were optimized,and the labeling efficacy was evaluated by direct determination.The internal references of DNA were divided into three groups and treated by various methods.The reference in group 1 was untreated,while that in group 2 was treated with bovine serum albumin and protease K.However,the reference in group 3 was treated by the method in group 2 and an additional procedure of DNA extraction.The sensitivities of above-mentioned samples were determined by dot blot with the labeled probe.Results The extraction effect of DNA from CHO cells by high salt method was satisfactory.The volumes for labeling was optimized as DNA 10 μg,Vial 4 16 μl and total volume 64 μl.The sensitivities of internal references in groups 1,2 and 3 were 1 pg,10 pg and 1 ng respectively.Conclusion The developed dot blot method with digoxin-labeled CHO cell DNA probe is stable and reliable,which may be used for determination of residual DNA content in CHO host cells in biologics.