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Peer-reviewedCC BY 4.0Molecules

Establishment of Reverse Transcription Polymerase Chain Reaction Method for Quantification of Residual Host Cell RNA in Recombinant Plasmid Drugs

  • Hua Bi1
  • Bo Zhang2
  • Xinchang Shi1
  • Xiang Li1
  • Dening Pei1
  • Shanshan Ma2
  • Huili Hou2
  • Ying Guo1
  • Youxue Ding1
  • Xi Qin1
  • Chenggang Liang1
  1. 1National Institutes for Drug Control, Beijing 100050, China
  2. 2Beijing Northland Biotechnology Co., Ltd., Beijing 100085, China
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Abstract

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.

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Cite

BibTeXRIS
BibTeX
@article{bi2026establishment,
  title = {{Establishment of Reverse Transcription Polymerase Chain Reaction Method for Quantification of Residual Host Cell RNA in Recombinant Plasmid Drugs}},
  author = {Hua Bi and Bo Zhang and Xinchang Shi and Xiang Li and Dening Pei and Shanshan Ma and Huili Hou and Ying Guo and Youxue Ding and Xi Qin and Chenggang Liang},
  journal = {Molecules},
  year = {2026},
  month = oct,
  doi = {10.3390/molecules31203670},
  url = {https://www.mdpi.com/1420-3049/31/20/3670},
  note = {CC BY 4.0},
}
RIS
TY  - JOUR
TI  - Establishment of Reverse Transcription Polymerase Chain Reaction Method for Quantification of Residual Host Cell RNA in Recombinant Plasmid Drugs
AU  - Hua Bi
AU  - Bo Zhang
AU  - Xinchang Shi
AU  - Xiang Li
AU  - Dening Pei
AU  - Shanshan Ma
AU  - Huili Hou
AU  - Ying Guo
AU  - Youxue Ding
AU  - Xi Qin
AU  - Chenggang Liang
PY  - 2026
DA  - 2026/10/01
JO  - Molecules
DO  - 10.3390/molecules31203670
UR  - https://www.mdpi.com/1420-3049/31/20/3670
AB  - 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.
SN  - 1420-3049
ER  -