Species-specific Virus Detection by qPCR
The selection of viruses required to be tested depends upon the origin and history of the production cell line, virus vaccine seed and raw material used in the manufacturing process. For example, PCR testing for the presence of human viruses is required when cells of human/primate origin or products obtained from human blood or tissues are involved. Examples where human virus PCR testing is advised, include human hybridomas, stem cells, and other cell lines such as Vero, HEK 293 and PER.C6 used in the production of Adenovirus vectors and vaccines. In cases where viruses cannot be readily grown in culture, PCR is currently the most effective tool to assess for contamination with such viruses. The human viruses that should be screened for are those associated with severe or oncogenic diseases and particularly those that might establish latent or abortive replication in cells. Manufacturers of certain influenza vaccines derived from cell culture are encouraged by the EU regulatory authorities to perform testing for specific respiratory viruses by PCR assays. Certain exogenous avian virus PCR assays are recommended if the influenza virus vaccine seed has historical culture within eggs, or a cell substrate is of avian origin. Due to the specificity of PCR, it is usual to perform multiple PCR assays in order to detect the full range of viruses of concern. The recommendation is that PCR assays when possible should detect several agents using degenerate or consensus primers provided the sensitivity of these assays is sufficient to assure product safety.

Vitrology’s real time qPCR assays contain a forward/reverse primer set and fluorogenic-probe to detect the presence of viral pathogen target sequences within test samples. Each virus testing qPCR package is customised for the client’s cell line to ensure they meet regulatory requirements. Each viral qPCR assay utilises TaqMan based probe technology which forms part of a customised high density qPCR array system fully compliant with the controls recommended in the regulatory guidelines.

The regulatory acceptance of highly sensitive virus detection methods for the testing of biopharmaceutical products has depended on PCR assays that are validated to ICH guidelines. All Vitrology’s assay validation, test design and assay performance control systems are carried out essentially as described in the regulatory guidelines, including US FDA, ICH Q2, European Phamacopoeia and as initially published by Lovatt et al., 2002. All assay validation parameter data including detection limit (DL), specificity, quantitative range, 95% cut-off and quantitation limit (QL) are described to our clients in our quality assurance approved validation reports.

The following control system is performed in compliance to European Pharmacopeia 2.6.21:

• Contamination control: Separate clean rooms for reagents, test samples, and positive controls. Each room and assay step contains a multi-step contamination control system.
• Nucleic Acid Extraction Interference: Prior to extraction each test sample is diluted and spiked with “extraction control DNA or RNA” that mimics low level virus genome nucleic acid.
• Nucleic Acid Amplification Interference: Prior to testing the sample and dilutions for the presence of “extraction control DNA or RNA” by qPCR each test sample is spiked with a secondary “amplification internal control nucleic acid ”.
• Optimisation of test sample volume: The results from the low level internal control qPCR assays determine the optimal sample concentration for testing with the viral target qPCR assays
• Positive control: Test sample is spiked with positive control viral nucleic acid at the detection limit to control for the presence of assay specific inhibitors.

Selected human and avian virus packages will be released in 2008.

Simian virus assays, and other species specific virus packages will be in development during 2008

*RNA viruses

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