Establishment of Reverse Transcription Polymerase Chain Reaction Method for Quantification of Residual Host Cell RNA...

The presence of residual host cell RNA in recombinant plasmid drugs poses potential safety concerns and is subject to strict regulatory limits. Therefore, reliable quantification methods are essential for quality control. This study aims to establish and validate a reverse transcription-quantitative polymerase chain reaction (RT-qPCR) method for the quantitative detection of residual Escherichia coli host cell RNA in recombinant plasmid drugs. The DNA digestion system was optimized, and three RNA reference materials from different sources (kit-supplied RNA, commercial Thermo Fisher RNA, and self-extracted RNA) were compared. The results for 2-, 4-, and 8-fold diluted samples were comparable. The three reference materials showed comparable electrophoretic band patterns and amplification curves, and they also yielded comparable RNA quantification results for the same samples. After systematic methodological validation of the established RT-qPCR method, it was applied to seven batches of recombinant plasmid drug substances and three batches of in-process intermediates. Notably, residual host cell RNA levels in all tested batches were below 0.2 μg/mg DNA, complying with current national and regional regulatory requirements.