Application of multivirus spike approach for viral clearance evaluation.

Viral contamination is a common risk to continuous cell line-derived biologics. Viral validation is thus required for license applications. Viral validation for chromatography procedures is routinely performed by spiking a model virus into the load material and performing the chromatography procedures at small scale under conditions equivalent to the commercial scale. With traditional cell-based infectivity assays, one can only spike one model virus at one time. Quantitative PCR methods (TaqMan) make it possible to spike multiple model viruses for a chromatography procedure simultaneously. TaqMan assays can quantify multiple types of viruses and other types of nucleic acid in a single sample without cross interference because of its extremely high specificity. Therefore, a multivirus spike approach was evaluated and compared to a single virus spike approach. The study was further extended to the evaluation of host cell DNA clearance. The data shows highly comparable viral and host cell DNA clearance between the single and multiple virus spike approaches. Application of a multivirus spike approach provides significant time, manpower, and cost savings for new drug development.

Detection of minute virus of mice using real time quantitative PCR in assessment of virus clearance during the purification of Mammalian cell substrate derived biotherapeutics.

A real time quantitative PCR assay has been developed for detecting minute virus of mice (MVM). This assay directly quantifies PCR product by monitoring the increase of fluorescence intensity emitted during enzymatic hydrolysis of an oligonucleotide probe labelled covalently with fluorescent reporting and quenching dyes via Taq polymerase 5'-->3' exonuclease activity. The quantity of MVM DNA molecules in the samples was determined using a known amount of MVM standard control DNA fragment cloned into a plasmid (pCR-MVM). We have demonstrated that MVM TaqMan PCR assay is approximately 1000-fold more sensitive than the microplate infectivity assay with the lowest detection limit of approximately one particle per reaction. The reliable detection range is within 100 to 10(9) molecules per reaction with high reproducibility. The intra assay variation is <2.5%, and the inter assays variation is <6.5% when samples contain >100 particles/assay. When we applied the TaqMan PCR to MVM clearance studies done by column chromatography or normal flow viral filtration, we found that the virus removal factors were similar to that of virus infectivity assay. It takes about a day to complete entire assay processes, thus, the TaqMan PCR assay is at least 10-fold faster than the infectivity assay. Therefore, we concluded that this fast, specific, sensitive, and robust assay could replace the infectivity assay for virus clearance evaluation.