Objective: To detect plasmid DNA residues in recombinant adeno-associated virus (rAAV) products by digital PCR (dPCR). Methods: Primer probes were designed for four different segments of the plasmid resistance gene KanR sequence, and singleplex and duplex dPCR were established to detect KanR residues. In duplex dPCR, double positive wells represent the sequence integrity between two sets of primer pairs, and the distribution of KanR sequences was assessed by testing the proportion of single and double positive wells. The presence of incorrectly packaged plasmid DNA within the viral capsid was evaluated by digesting free plasmid DNA with DNase Ⅰ. Results: The KanR residues measured in four groups by single dPCR were basically consistent (1.054×108-1.467×108) copies·μL-1. There was no significant difference between the measured results of duplex dPCR and singleplex dPCR in each channel. It showed good linearity (r＞0.999) and repeatability (RSD<20%) in 6-6 000 copies·μL-1 concentration range for the established duplex dPCR. The proportion of double positive wells in the dual PCR of 1/2, 2/3, and 3/43 combinations was basically consistent, indicating that the breakage of the KanR gene may tend to be more random. The results before and after DNase Ⅰ treatment suggested that the proportion of large fragments of plasmid DNA (1/4 double positive) that are wrongly packaged was higher than that of free plasmid DNA. Conclusion: The dPCR technology can be used to detect plasmid DNA residues in rAAV products, and multiple dPCR can also evaluate the size and distribution of plasmid DNA fragments.
FAN Wen-chao, YU Lei, AN Yi-fang, WANG Guang-yu, SHI Xin-chang, ZHOU Yong
. Detection of plasmid DNA residues in rAAV products by digital PCR technology *[J]. Chinese Journal of Pharmaceutical Analysis, 2023
, 43(12)
: 2004
-2009
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DOI: 10.16155/j.0254-1793.2023.12.02
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