Development And NDA-Level Validation Of A Real-Time PCR Procedure

Development and NDA-Level Validation of a Real-Time PCR Procedure for Detection and Quantification of Residual E.coli DNA Contamination of Biopharmaceutical Products

Author: Dan Papa, Pedro J. Morales, and Michael D. Sadick

Escherichia coli (E. coli) has been commonly used for the production of biopharmaceuticals. Among the impurities that must be monitored in biopharmaceuticals is residual host-cell DNA (HCD). This study describes the development and subsequent NDA-level validation of a real time PCR procedure developed in response to a client’s need to improve the sensitivity of detection and quantification of residual E. coli HCD in their drug product, previously determined via total DNA threshold assay. This approach involved the design of a primer set capable of priming and amplifying a very short sequence (122 bp) inside the 16S rRNA – Glu-tRNA region of rrnB operon of E. coli, using TaqMan chemistry. Based on preliminary end-point PCR tests, the visual detection limit was 0.001 pg genomic DNA (gDNA). The procedure was successfully developed, optimized and validated for use in commercial drug product release. The limit of detection (LOD) was calculated to be 0.050 pg gDNA, whereas the limit of quantification was calculated to be 0.500 pg gDNA. The assay showed accuracy and precision (both repeatability and intermediate precision) with DNA content of even 10pg and below. Thus, the real-time PCR procedure has proven to be a very sensitive and powerful tool for cGMP quantitative detection of E. coli HCD.