Applied research on qPCR for determination of large fragment host cell residual DNA in recombinant adeno-associated virus products
Objective To investigate the feasibility of using quantitative PCR(qPCR)technology to detect large fragments of host cell residual DNA(HCD)in recombinant adeno-associated virus(rAAV)gene therapy products.Methods Four different serotypes of rAAV were extracted for the nucleic acids,two fragment sequences of 244 bp and 562 bp within the long terminal repeat sequence(LTR)in the genome of host cells HEK293 were specifically quantified by qPCR,and the proportion of HCD in the total nucleic acids was calculated.Results Large fragments of HCD in qPCR quantifiable range were detected in four different serotype rAAV products,with the abundance ranging from 0.3％to 5.4％.As the length of the detected fragment increased,the abundance of HCD fragments showed a decreasing trend.Conclusion qPCR technology can be used to determine the presence of large fragments of HCD in rAAV products.
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