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Evaluation of viral removal by nanofiltration using real‐time quantitative polymerase chain reaction
Author(s) -
Zhao Xiaowen,
Bailey Mark R.,
Emery Warren R.,
Lambooy Peter K.,
Chen Dayue
Publication year - 2007
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1042/ba20060195
Subject(s) - nanofiltration , nuclease , infectivity , virus , rna , chemistry , virology , murine leukemia virus , real time polymerase chain reaction , nucleic acid , chromatography , biology , dna , biochemistry , membrane , gene
Nanofiltration is commonly introduced into purification processes of biologics produced in mammalian cells to serve as a designated step for removal of potential exogenous viral contaminants and endogenous retrovirus‐like particles. The LRV (log reduction value) achieved by nanofiltration is often determined by cell‐based infectivity assay, which is time‐consuming and labour‐intensive. We have explored the possibility of employing QPCR (quantitative PCR) to evaluate LRV achieved by nanofiltration in scaled‐down studies using two model viruses, namely xenotropic murine leukemia virus and murine minute virus. We report here the successful development of a QPCR‐based method suitable for quantification of virus removal by nanofiltration. The method includes a nuclease treatment step to remove free viral nucleic acids, while viral genome associated with intact virus particles is shielded from the nuclease. In addition, HIV Armored RNA® was included as an internal control to ensure the accuracy and reliability of the method. The QPCRbased method described here provides several advantages such as better sensitivity, faster turnaround time, reduced cost and higher throughput over the traditional cell‐based infectivity assays.