High-Throughput and Quantitative Detection of Residual NS0 and CHO Host Cell Genomic DNA

LEVEL: INTERMEDIATE R esidual host cell DNA removal in the processing of biological pharmaceuticals is an important metric for consistency of the purification process. There are also safety concerns with respect to the oncogenic potential of residual DNA from continuous cell lines. The acceptable residual amount of DNA is 10 ng/dose for parentally administrated drugs produced from continuous cell lines, based on WHO (World Health Organization) guidelines (1). Traditional methods to evaluate residual DNA level during process development, such as the Threshold assay, are expensive, low throughput, and labor intensive (Molecular Devices Corporation Threshold total DNA assay system, www.moleculardevices.com). To provide quick and highthroughput evaluation of the DNA removal process, we developed TaqMan qPCR (quantitative polymerase chain reaction) assays coupled with 96-well DNA extraction methodology. To meet the sensitivity requirements, we required that the TaqMan target sequence for the hosts be at least 100 base pairs (bp) long, present in multiple copies, and interspersed throughout the host genome. Our expectation was that dispersed target sequences with high occurrence in the genome would yield assays of greater sensitivity and guard against the possibility of nondetection of genome portions lacking the targeted sequence. Two TaqMan assays (B1 and R-repeat assays) were developed for detection of genomic DNA of mouse cells (NS0), and three (CHO-49, CHO-63 and CHO G-repeat assays) were designed for detection of genomic DNA of Chinese hamster ovary cells (CHO). An additional TaqMan assay was designed to target luciferase sequence carried by a pGL3-Basic vector, which served as a spike-control to evaluate host cell DNA extraction and TaqMan quantitation. The 96-well extraction conditions were optimized through statistical experimental design. TaqMan assays showed good correlation with the Threshold assay when evaluating samples from actual purification process streams. The R2 is 0.9843 in log scale of DNA, with 95% confidence intervals obtained from the replicated assays. EXPERIMENTAL: MATERIALS AND METHODS TaqMan PCR: The primer-probe sequence was designed using Applied Biosystems’ Primer Express software (www.appliedbiosystems.com). All probes were TAMRA quenched, 5’ -VIC labeled (except CHO49-probe with 6-FAM at its 5’) and acquired from Applied Biosystems. Two primer-probe sets, referred to as B1 and R-repeat assays, were designed for detection of mouse genomic DNA (2, 3). Three primer-probe sets were designed for detection of CHO DNA. Two sets, referred to as CHO49 and CHO63, were designed against CHO Alu-equivalent repeats, the sequence of clone 49C and Clone 63, respectively (4); and the third, CHO-G repeat, was designed against the novel hamster G-repeat sequence (5). The primer-probes sequence (5’– 3’) is listed in the “Sequences” box. WWW.PHOTOS.COM