Comparison of two pretreatment methods for detection of free and mispackaged host cell DNA in recombinant adeno-associated virus
Objective To compare the application of two pretreatment methods,deoxyribonuclease(DNase)treatment and ligand affinity,in detecting the residual amount of free and mispackaged host cell DNA(HCDNA)in recombinant adeno-associated virus(rAAV).Methods Free and mispackaged HCDNA were isolated by DNase treatment and ligand affinity respectively,and then the nucleic acid was extracted and detected by qPCR.The accuracy and reproducibility of two pretreatment methods for detecting HCDNA residues were compared.Results Using DNase treatment,the result of nucleic acid quantitative detection in non-DNase-treated group was the total residual amount of HCDNA,that in DNase-treated group was the amount of mispackaged HCDNA,and the difference between them was the residual amount of free HCDNA.The recovery rates of both the untreated and treated groups were more than 75％,and the RSD of reproducibility was less than 30％.Using affinity extraction method,with the affinity ligand combined with rAAV,the result of recovery rate of mispackaged HCDNA was over 75％,and that of free HCDNA was only 36％.Conclusion DNase treatment method can effectively detect free and mispackaged HCDNA,laying a foundation for further research.
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