Multiplex RT Q-PCR assay for simultaneous quantification of three viruses used for validation of virus clearance by biopharmaceutical production |
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Authors: | Scott Lute Hua Wang Davonie Sanchez Janet Barletta Qi Chen Kurt Brorson |
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Institution: | aCenter for Drug Evaluation and Research, Food and Drug Administration, 10903 New Hampshire Ave., Silver Spring, MD 20993, USA;bLate Stage Purification, Process Research and Development, Genentech Inc., One DNA Way, South San Francisco, CA 94080, USA |
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Abstract: | Virus removal studies are used to insure the safety of biopharmaceutical products by quantitatively estimating the viral clearance capacity by the manufacturing process. Virus quantification assays are used to measure the log10 clearance factor of individual purification unit operations in spike recovery studies. We have developed a multiplex RT Q-PCR assay that detects and quantifies three commonly used model viruses X-MuLV, SV40, and MMV simultaneously. This RT Q-PCR multiplex assay has a 6 log10 dynamic range with a limit of detection (LOD) of ≈1 genome copy/μL. Amplification profiles are similar to existing singleplex assays. Overall, this RT Q-PCR multiplex assay is highly quantitative, accurately identifies multiple viruses simultaneously, and may prove useful to validate viral clearance of biological products in small scale studies. |
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Keywords: | Q-PCR Viral safety Monoclonal antibodies |
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