Digital droplet PCR for influenza vaccine development

Development of influenza vaccine processes requires virus quantification to optimize conditions in cell culture or in the associated downstream purification steps. Modern methods include qPCR, which utilizes TaqMan chemistry to detect and quantify viral RNA by comparison of a RNA standard of known concentration. Digital droplet PCR (ddPCR) is similar to qPCR in that it shares the same chemistry for nucleic acid detection. However, in ddPCR, the sample is diluted into partitions ('droplets') in order to separate and isolate single molecules. Upon PCR amplification, the droplet's fluorescent intensity depends on the presence or absence of the target; as such, positive and negative droplets are identified, which allows for absolute quantification of the viral genomes. The digital approach has enabled several key advantages. First, a standard is no longer required. Second, efficiency of the reverse transcription and the kinetics of the amplification, principles in qPCR, have no impact on the final digital PCR quantification. For this reason, the extracted RNA does not need to be purified from the reagents needed to lyse the virus. Also, viral associated RNA released by infected cells can be measured directly, further improving the quality of the data generated. Additional improvements to the approach include duplexing with a second assay that measures host cell DNA concentration. The method has been successfully implemented with automation in support of multiple upstream and downstream process development efforts for influenza vaccine manufacturing. (C) 2015 Published by Elsevier B.V.