Applications of quantitative PCR in the biosafety and genetic stability assessment of biotechnology products

Abstract

High throughput screening, increased accuracy and the coupling of real-time quantitative PCR (Q-PCR) to robotic set-up systems are beginning to revolutionise biotechnology. Applications of Q-PCR within biotechnology are discussed with particular emphasis on the following areas of biosafety and genetic stability testing: (a) determination of the biodistribution of gene therapy vectors in animals; (b) quantification of the residual DNA in final product therapeutics; (c) detection of viral and bacterial nucleic acid in contaminated cell banks and final products; (d) quantification of the level of virus removal in process validation viral clearance studies; (e) specific detection of retroviral RT activity in vaccines with high sensitivity; and (f) transgene copy number determination for monitoring genetic stability during production. Methods employed for Q-PCR assay validation as required in ICH Topic Q2A Validation of Analytical Methods: Definitions and Terminology (1st June 1995) are also reviewed.

Fig. 1

The 5′ Exonuclease Assay and the Real-Time Q-PCR Amplification Plot. (a) Diagram showing cleavage of the FAM reporter dye on the TaqMan probe during PCR amplification. (b) Amplification plot showing exponential increase in FAM reporter dye and the calculation of the C_T value.

Fig. 2

Standard curve showing linear relationship between C_T and starting copy number. Serial 10-fold dilutions in the range of 10⁶–10 copies of viral target DNA were analysed as standards. The slope, y-intercept and correlation co-efficient of the generated standard curve are shown on the right hand side.

Fig. 3

5′–3′ multiple sequence alignment of a conserved region of the Bovine circovirus and Porcine circovirus rep region. TaqMan primers and probe are designed as degenerated oligonucleotides. Degenerated nucleotides are: Y=CT; R=A+G; S=G+C; W=A+T.

Fig. 4

Overview of the F-PERT assay.

Fig. 5

Q-PCR for viral clearance studies. For example, X-MLV stock of 10⁹ RNA genome copies per ml (approx. 10⁶ TCID₅₀) is diluted 10% in the load to give a 10⁸ X-MLV genome per ml spiked load. If the background signal from the bulk harvest material is negligible and the quantification limit of the X-MLV Q-PCR is 10⁴ RNA copies per ml, we have a 4-log window to calculate viral RNA removal.