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Quantitative detection of residual porcine host cell DNA by real-time PCR
Jen-Ting Chang1, Yu-Chen Chen1, Yu-Chi Chou1
1Animal Technology Institute, Chunan, Miaoli, Taiwan.
Abstract:
All biological products are derived from complex living systems and are often mixed with large numbers of impurities. For reasons of safety, residual host-cell DNA must be eliminated during processing. To assay host-cell DNA content in biopharmaceutical products derived from porcine sources, this study applies the quantitative real-time polymerase chain reaction (Q-PCR) method. The optimized assay in this study is based on the pol region of the porcine endogenous retrovirus (PERV). Assay validation results demonstrate that the proposed assay has appropriate accuracy, preciseness, reproducibility, and sensitivity. Primer and probe specificity are evaluated in real-time Q-PCR reactions using genomic DNA from rabbit, mouse, cat, hamster, monkey, human cell, yeast, and Escherichia coli as templates. The sensitivity of real-time Q-PCR is determined using genomic DNA from the porcine kidney cell line. The reliable detection range is within 0.5-10(5) pg/reaction. The limit of quantitation is 500 fg. The sensitivity of the assay meets the authority criterion. Moreover, the assay is applied to determine the level of host-cell DNA in recombinant human coagulation factor IX (rhFIX) from transgenic pigs. The real-time Q-PCR assay is thus a promising new tool for quantitative detection and clearance validation of residual porcine DNA when manufacturing recombinant therapeutics.
Insights
A new quantitative real-time PCR assay effectively measures residual porcine DNA in biopharmaceuticals. This method ensures product safety by accurately quantifying host-cell DNA from transgenic pigs.
Area of Science:
- Biotechnology
- Molecular Biology
- Analytical Chemistry
Background:
- Biological products often contain impurities, including residual host-cell DNA, necessitating strict safety controls.
- Eliminating host-cell DNA is critical for the safety of biopharmaceutical products, especially those derived from animal sources.
- Porcine endogenous retrovirus (PERV) DNA is a key impurity to monitor in products from transgenic pigs.
Purpose of the Study:
- To develop and validate a sensitive quantitative real-time PCR (Q-PCR) assay for detecting residual porcine DNA.
- To establish an accurate method for quantifying host-cell DNA in biopharmaceutical products derived from porcine sources.
- To validate the assay's performance for safety and regulatory compliance in biomanufacturing.
Main Methods:
- Quantitative real-time polymerase chain reaction (Q-PCR) targeting the pol region of porcine endogenous retrovirus (PERV).
- Assay validation including accuracy, precision, reproducibility, and sensitivity testing.
- Specificity evaluation using genomic DNA from various species and microorganisms.
Main Results:
- The developed Q-PCR assay demonstrated high accuracy, precision, reproducibility, and sensitivity.
- The assay reliably detects porcine DNA within a range of 0.5-10^5 pg/reaction, with a limit of quantitation at 500 fg.
- Specificity was confirmed against DNA from non-porcine sources, including human, rabbit, mouse, and E. coli.
- The assay successfully quantified residual porcine DNA in recombinant human coagulation factor IX (rhFIX) from transgenic pigs.
Conclusions:
- The optimized Q-PCR assay provides a reliable and sensitive tool for quantifying residual porcine DNA.
- This method is suitable for clearance validation and safety assessment of biopharmaceuticals derived from transgenic pigs.
- The assay meets regulatory sensitivity criteria and supports the manufacturing of safe and effective recombinant therapeutics.
